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1.  Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region 
Biochemical pharmacology  2012;84(5):722-735.
The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)n repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)n was n = 4>5≫6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.
PMCID: PMC3965201  PMID: 22728919
aryl hydrocarbon receptor; long-term estrogen exposure; epigenetic; (GGGGC)n repeat polymorphism; guanine-quadruplex; 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
2.  Anorectal atresia and variants at predicted regulatory sites in candidate genes 
Annals of human genetics  2012;77(1):31-46.
Anorectal atresia is a serious birth defect of largely unknown etiology but candidate genes have been identified in animal studies and human syndromes. Because alterations in the activity of these genes might lead to anorectal atresia, we selected 71 common variants predicted to be in transcription factor binding sites, CpG windows, splice sites, and miRNA target sites of 25 candidate genes, and tested for their association with anorectal atresia. The study population comprised 150 anorectal atresia cases and 623 control infants without major malformations. Variants predicted to affect transcription factor binding, splicing, and DNA methylation in WNT3A, PCSK5, TCF4, MKKS, GLI2, HOXD12, and BMP4 were associated with anorectal atresia based on a nominal P value <0.05. The GLI2 and BMP4 variants are reported to be moderately associated with gene expression changes (Spearman’s rank correlation coefficients between −0.260 and 0.226). We did not find evidence for interaction between maternal pre-pregnancy obesity and variants in MKKS, a gene previously associated with obesity, on the risk of anorectal atresia. Our results for MKKS support previously suggested associations with anorectal malformations. Our findings suggest that more research is needed to determine whether altered GLI2 and BMP4 expression is important in anorectal atresia in humans.
PMCID: PMC3535506  PMID: 23127126
anorectal malformations; imperforate anus; hindgut; congenital abnormalities
3.  Evaluation of Genes Involved in Limb Development, Angiogenesis, and Coagulation as Risk Factors for Congenital Limb Deficiencies 
We conducted a population-based case-control study of single nucleotide polymorphisms (SNPs) in selected genes to find common variants that play a role in the etiology of limb deficiencies (LD)s. Included in the study were 389 infants with LDs of unknown cause and 980 unaffected controls selected from all births in New York State (NYS) for the years 1998 to 2005. We used cases identified from the NYS Department of Health (DOH) Congenital Malformations Registry. Genotypes were obtained for 132 SNPs in genes involved in limb development (SHH, WNT7A, FGF4, FGF8, FGF10, TBX3, TBX5, SALL4, GREM1, GDF5, CTNNB1, EN1, CYP26A1, CYP26B1), angiogenesis (VEGFA, HIF1A, NOS3), and coagulation (F2, F5, MTHFR). Genotype call rates were >97% and SNPs were tested for departure from Hardy-Weinberg expectations by race/ethnic subgroups. For each SNP, odds ratios (OR)s and confidence intervals (CI)s were estimated and corrected for multiple comparisons for all LDs combined and for LD subtypes. Among non-Hispanic white infants, associations between FGF10 SNPs rs10805683 and rs13170645 and all LDs combined were statistically significant following correction for multiple testing (OR=1.99; 95% CI=1.43-2.77; uncorrected p=0.000043 for rs10805683 heterozygous genotype, and OR=2.37; 95% CI=1.48-3.78; uncorrected p=0.00032 for rs13170645 homozygous minor genotype). We also observed suggestive evidence for associations with SNPs in other genes including CYP26B1 and WNT7A. Animal studies have shown that FGF10 induces formation of the apical ectodermal ridge and is necessary for limb development. Our data suggest that common variants in FGF10 increase the risk for a wide range of non-syndromic limb deficiencies.
PMCID: PMC3448837  PMID: 22965740
limb deficiencies; polymorphisms; FGF10
4.  A genome-wide association study identifies susceptibility loci for non-syndromic sagittal craniosynostosis near BMP2 and within BBS9 
Nature genetics  2012;44(12):1360-1364.
Sagittal craniosynostosis is the most common form of craniosynostosis, affecting approximately one of 5,000 newborns. We conducted the first genome-wide association study (GWAS) for non-syndromic sagittal craniosynostosis (sNSC) using 130 non-Hispanic white (NHW) case-parent trios. Robust associations were observed in a 120 kb region downstream of BMP2, flanked by rs1884302 (P = 1.13 × 10−14; odds ratio [OR] = 4.58) and rs6140226 (P = 3.40 × 10−11; OR = 0.24) and within a 167 kb region of BBS9 between rs10262453 (P = 1.61 × 10−10; OR=0.19) and rs17724206 (P = 1.50 × 10−8; OR = 0.22). We replicated the associations to both loci [rs1884302 (P = 4.39 × 10−31); rs10262453 (P = 3.50 × 10−14)] in an independent NHW population of 172 unrelated sNSC probands and 548 controls. Both BMP2 and BBS9 are genes with a role in skeletal development warranting functional studies to further understand the etiology of sNSC.
PMCID: PMC3736322  PMID: 23160099
genome-wide association study; non-syndromic sagittal craniosynostosis; BMP2; BBS9; meta-analysis; nonsyndromic
5.  Hirschsprung’s disease and variants in genes that regulate enteric neural crest cell proliferation, migration and differentiation 
Journal of human genetics  2012;57(8):485-493.
Hirschsprung’s disease (HSCR) results from failed colonization of the embryonic gut by enteric neural crest cells (ENCCs); colonization requires RET proto-oncogene (RET) signaling. We sequenced RET to identify coding and splice-site variants in a population-based case group and we tested for associations between HSCR and common variants in RET and candidate genes (ASCL1, HOXB5, L1CAM, PHOX2B, PROK1, PROKR1) chosen because they are involved in ENCC proliferation, migration, and differentiation in animal models. We conducted a nested case-control study of 304 HSCR cases and 1 215 controls. Among 38 (12.5%) cases with 34 RET coding and splice-site variants, 18 variants were previously unreported. We confirmed associations with common variants in HOXB5 and PHOX2B but the associations with variants in ASCL1, L1CAM, and PROK1 were not significant after multiple comparisons adjustment. RET variants were strongly associated with HSCR (P values between 10−3 and 10−31) but this differed by race/ethnicity: associations were absent in African-Americans. Our population-based study not only identified novel RET variants in HSCR cases, it showed that common RET variants may not contribute to HSCR in all race/ethnic groups. The findings for HOXB5 and PHOX2B provide supportive evidence that genes regulating ENCC proliferation, migration, and differentiation could be risk factors for HSCR.
PMCID: PMC3503526  PMID: 22648184
congenital abnormalities; enteric nervous system; Hirschsprung disease; RET
6.  Lysosomal storage disorder 4+1 multiplex assay for newborn screening using tandem mass spectrometry: Application to a small-scale population study for five lysosomal storage disorders 
We sought to modify a previously published tandem mass spectrometry method of screening for 5 lysosomal storage disorders (LSDs) in order to make it better suited for high-throughput newborn screening.
Two 3-mm dried blood spot (DBS) punches were incubated, each with a different assay solution. The quadruplex solution was used for screening for Gaucher, Pompe, Krabbe and Fabry diseases, while a separate solution was used for Niemann–Pick A/B disease.
The mean activities of acid-β-glucocerebrosidase (ABG), acid sphingomyelinase (ASM), acid glucosidase (GAA), galactocerebroside-β-galactosidase (GALC) and acid-galactosidase A (GLA) were measured on 5055 unidentified newborns. The mean activities (compared with their disease controls) were, 15.1 (0.35), 22.2 (1.34), 16.8 (0.51), 3.61 (0.23), and 20.7 (1.43) (μmol/L/h), respectively. The number of specimens that fell below our retest level cutoff of <20% daily mean activity (DMA) for each analyte is: ABG (6), ASM (0), GAA (5), GALC (17), and GLA (2).
This method provides a simplified and reliable assay for screening for five LSDs with clear distinction between activities from normal and disease samples. Advantages of this new method include significant decreases in processing time and the number of required assay solutions and overall decreased complexity.
PMCID: PMC3443687  PMID: 22548856
Newborn screening; Lysosomal storage disorder; Multiplex; Tandem mass spectrometry
7.  Folate and Vitamin B12 Related Genes and Risk for Omphalocele 
Human Genetics  2011;131(5):739-746.
Both taking folic acid-containing vitamins around conception and consuming food fortified with folic acid have been reported to reduce omphalocele rates. Genetic factors are etiologically important in omphalocele as well; our pilot study showed a relationship with the folate metabolic enzyme gene methylenetetrahydrofolate reductase (MTHFR). We studied 169 non-aneuploid omphalocele cases and 761 unaffected, matched controls from all New York State births occurring between 1998 and 2005 to look for associations with single nucleotide polymorphisms (SNPs) known to be important in folate, vitamin B12, or choline metabolism. In the total study population, variants in the transcobalamin receptor gene (TCblR), rs2232775 (Q8R), and the MTHFR gene, rs1801131 (1298A>C), were significantly associated with omphalocele. In African-Americans significant associations were found with SNPs in genes for the vitamin B12 transporter (TCN2) and the vitamin B12 receptor (TCblR). A SNP in the homocysteine-related gene, betaine-homocysteine S-methyltransferase (BHMT), rs3733890 (R239Q), was significantly associated with omphalocele in both African-Americans and Asians. Only the TCblR association in the total population remained statistically significant if Bonferroni correction was applied. The finding that transcobalamin receptor (TCblR) and transporter (TCN2) SNPs and a BHMT SNP were associated with omphalocele suggests that disruption of methylation reactions, in which folate, vitamin B12, and homocysteine play critical parts, may be a risk factor for omphalocele. Our data, if confirmed, suggest that supplements containing both folic acid and vitamin B12 may be beneficial in preventing omphaloceles.
PMCID: PMC3374579  PMID: 22116453
omphalocele; folate; vitamin B12; homocysteine; transcobalamin; transcobalamin receptor
8.  Newborn screening and early biochemical follow-up in combined methylmalonic aciduria and homocystinuria, cblC type, and utility of methionine as a secondary screening analyte 
Molecular genetics and metabolism  2009;99(2):116-123.
Combined methylmalonic aciduria and homocystinuria, cobalamin C (cblC) type, is an inherited disorder of vitamin B12 metabolism caused by mutations in MMACHC. CblC typically presents in the neonatal period with neurological deterioration, failure to thrive, cytopenias, and multisystem pathology including renal and hepatic dysfunction. Rarely, affected individuals present in adulthood with gait ataxia and cognitive decline. Treatment with hydroxycobalamin may ameliorate the clinical features of early-onset disease and prevent clinical late-onset disease. Propionic acidemia (PA), methylmalonic acidemia (MMA), and various disorders of cobalamin metabolism are characterized by elevated propionylcarnitine (C3) on Newborn Screening (NBS). Distinctions can be made between these disorders with secondary analyte testing. Elevated methionine is already routinely used as a NBS marker for cystathionine ß-synthase deficiency. We propose that low methionine may be useful as a secondary analyte for specific detection of cbl disorders among a larger pool of infants with elevated C3 on NBS.
Retrospective analysis of dried blood spot (DBS) data in patients with molecularly confirmed cblC disease.
9 out of 10 patients with confirmed cblC born in New York between 2005 and 2008 had methionine below 13.4 μmol/L on NBS. Elevated C3, elevated C3:C2 ratio, and low methionine were incorporated into a simple screening algorithm that can be used to improve the specificity of newborn screening programs and provide a specific and novel method of distinguishing cblC from other disorders of propionate metabolism prior to recall for confirmatory testing.
It is anticipated that this algorithm will aid in early and specific detection of cobalamin C, D, and F diseases, with no additional expense to NBS laboratories screening for organic acidemias and classical homocystinuria.
PMCID: PMC2914534  PMID: 19836982
cblC; cobalamin; methylmalonic aciduria; methionine; newborn screening; propionylcarnitine

Results 1-8 (8)