Folate and vitamin B12 status and predicted neural tube defects risk among nonpregnant women of reproductive age from the Malawi National Micronutrient Survey, 2015-2016.

BACKGROUND: Maternal folate and vitamin B12 deficiency can lead to serious adverse pregnancy outcomes. There are no nationally representative estimates on folate and vitamin B12 status among women of reproductive age (WRA) in Malawi. OBJECTIVE: We assessed folate and vitamin B12 status among nonpregnant WRA in Malawi and predicted the risk of folate-sensitive neural tube defects (NTDs) were they to become pregnant. METHODS: Using data from the cross-sectional, nationally representative 2015-2016 Malawi Micronutrient Survey, we calculated the proportion of folate and vitamin B12 deficiency and insufficiency by demographic characteristics among 778 nonpregnant WRA (15-49 years). We predicted NTD prevalence using red blood cell (RBC) folate distributions and a published Bayesian model of the association between RBC folate and NTD risk. Analyses accounted for complex survey design. RESULTS: Among WRA, 8.5% (95% CI: 6.2, 11.6) and 13.3% (10.0, 17.4) had serum (<7 nmol/L) and RBC folate (<305 nmol/L) deficiency, respectively. The proportion of vitamin B12 deficiency (<148 pmol/L) and insufficiency (≤221 pmol/L) was 11.8% (8.6, 16.0) and 40.6% (34.1, 47.4), respectively. RBC folate insufficiency (<748 nmol/L, defined as the concentration associated with the threshold for elevated NTD risk: >8 cases per 10,000 births) was widespread: 81.4% (75.0, 86.4). The predicted NTD risk nationally was 24.7 cases per 10,000 live births. RBC folate insufficiency and higher predicted NTD risk were more common among WRA living in urban areas or with higher education. CONCLUSIONS: These findings highlight the importance of nutritional and NTD surveillance in Malawi and the opportunity for improving folate and vitamin B12 nutrition among Malawian WRA.

Folate and vitamin B12 usual intake and biomarker status by intake source in United States adults aged ≥19 y: NHANES 2007-2018.

BACKGROUND: Folate and vitamin B12 are important biomarkers of nutritional status of populations. OBJECTIVES: This study aims to estimate folate and vitamin B12 usual intakes among United States adults and examine folate and vitamin B12 biomarker status by intake source. METHODS: We analyzed data for United States adults aged ≥19 y from National Health and Nutrition Examination Survey 2007-2018 (n = 31,128), during which time voluntary corn masa flour (CMF) fortification was started. Usual intake was estimated using the National Cancer Institute method. Folate intake included folate from natural foods and folic acid from 4 sources: enriched cereal grain products (ECGPs), CMF, ready-to-eat cereals (RTEs), and folic acid-containing supplements (SUP). Vitamin B12 intake was mainly from food and supplements. RESULTS: The median natural food folate intake (222 µg dietary folate equivalents (DFE)/d) was below the estimated average requirement (EAR) of 320 µg DFE/d. The proportions of those who consumed folic acid from ECGP/CMF only, ECGP/CMF + RTE, ECGP/CMF + SUP, and ECGP/CMF + RTE + SUP were 50%, 18%, 22%, and 10%, respectively. Median usual folic acid intakes (µg/d) were 236 (IQR: 152, 439) overall and 134, 313, 496, and 695 in the ECGP/CMF only, ECGP/CMF + RTE, ECGP/CMF + SUP, and ECGP/CMF + RTE + SUP folic acid consumption groups, respectively. Overall, 2.0% (95% CI: 1.7%, 2.3%) of adults, all of whom used folic acid supplements, consumed greater than the tolerable upper intake level (UL) of 1000 µg/d folic acid. The median usual vitamin B12 intake (µg/d) was 5.2 for vitamin B12 supplement nonusers and 21.8 for users. Consumption of RTE and/or supplements with folic acid was associated with higher serum and red blood cell folate concentrations. Vitamin B12 supplement users had significantly higher serum vitamin B12 concentrations. CONCLUSIONS: Folic acid fortification plays a critical role in helping United States adults meet the folate EAR. At current fortification levels, United States adults who do not consume supplements do not have the usual folic acid intake exceeding the UL.

Estimating the serum folate concentration that corresponds to the red blood cell folate concentration threshold associated with optimal neural tube defects prevention: A population-based biomarker survey in Southern India.

BACKGROUND: RBC folate concentrations are monitored at the population level, with a recommended threshold for optimal neural tube defect (NTD) prevention. A corresponding threshold for serum folate has not been established. OBJECTIVES: This study aimed to estimate the serum folate insufficiency threshold corresponding to the RBC folate threshold for NTD prevention and examine how this threshold is modified by vitamin B12 status. METHODS: Participants were women (15-40 y; not pregnant or lactating; n = 977) from a population-based biomarker survey in Southern India. RBC folate and serum folate were measured via microbiologic assay. RBC folate deficiency (<305 nmol/L) and insufficiency (<748 nmol/L), serum vitamin B12 deficiency (<148 pmol/L) and vitamin B12 insufficiency (<221 pmol/L), elevated plasma MMA (>0.26 µmol/L), elevated plasma homocysteine (>10.0 µmol/L), and elevated HbA1c (≥6.5%) were evaluated. Bayesian linear models were used to estimate unadjusted and adjusted thresholds. RESULTS: Compared with adequate vitamin B12 status, the estimated serum folate threshold was higher in participants with serum vitamin B12 deficiency (72.5 vs. 28.1 nmol/L) or vitamin B12 insufficiency (48.7 vs. 24.3 nmol/L) and elevated MMA (55.6 vs. 25.9 nmol/L). The threshold was lower in participants with elevated HbA1c (HbA1c ≥6.5% vs. <6.5%; 21.0 vs. 40.5 nmol/L). CONCLUSIONS: The estimated serum folate threshold for optimal NTD prevention was similar to previous reports (24.3 vs. 25.6 nmol/L) among participants with sufficient vitamin B12 status. However, this threshold was more than 2-fold higher in participants with vitamin B12 deficiency and substantially higher across all indicators of insufficient vitamin B12 status (<221 pmol/L, elevated MMA, combined B12, impaired vitamin B12 status), and lower in participants with elevated HbA1c. Findings suggest a serum folate threshold for NTD prevention may be possible in some settings; however, it may not be appropriate in populations with high prevalence of vitamin B12 insufficiency. Am J Clin Nutr 2023;xx:xx-xx. This trial was registered at https://clinicaltrials.gov as NCT04048330.

Folic acid supplementation and malaria susceptibility and severity among people taking antifolate antimalarial drugs in endemic areas.

BACKGROUND: Description of the condition Malaria, an infectious disease transmitted by the bite of female mosquitoes from several Anopheles species, occurs in 87 countries with ongoing transmission (WHO 2020). The World Health Organization (WHO) estimated that, in 2019, approximately 229 million cases of malaria occurred worldwide, with 94% occurring in the WHO's African region (WHO 2020). Of these malaria cases, an estimated 409,000 deaths occurred globally, with 67% occurring in children under five years of age (WHO 2020). Malaria also negatively impacts the health of women during pregnancy, childbirth, and the postnatal period (WHO 2020). Sulfadoxine/pyrimethamine (SP), an antifolate antimalarial, has been widely used across sub-Saharan Africa as the first-line treatment for uncomplicated malaria sTo examine the effects of folic acid supplementation, at various doses, on malaria susceptibility (risk of infection) and severity among people living in areas with various degrees of malaria endemicity. We will examine the interaction between folic acid supplements and antifolate antimalarial drugs. Specifically, we will aim to answer the following. Among uninfected people living in malaria endemic areas, who are taking or not taking antifolate antimalarials for malaria prophylaxis, does taking a folic acid-containing supplement increase susceptibility to or severity of malaria infection? Among people with malaria infection who are being treated with antifolate antimalarials, does folic acid supplementation increase the risk of treatment failure?Criteria for considering studies for this review Types of studies Inclusion criteria Randomized controlled trials (RCTs) Quasi-RCTs with randomization at the individual or cluster level conducted in malaria-endemic areas (areas with ongoing, local malaria transmission, including areas approaching elimination, as listed in the World Malaria Report 2020) (WHO 2020) Exclusion criteria Ecological studies Observational studies In vivo/in vitro studies Economic studies Systematic literature reviews and meta-analyses (relevant systematic literature reviews and meta-analyses will be excluded but flagged for grey literature screening) Types of participants Inclusion criteria Individuals of any age or gender, living in a malaria endemic area, who are taking antifolate antimalarial medications (inclu

Folic Acid and the Prevention of Birth Defects: 30 Years of Opportunity and Controversies.

For three decades, the US Public Health Service has recommended that all persons capable of becoming pregnant consume 400 µg/day of folic acid (FA) to prevent neural tube defects (NTDs). The neural tube forms by 28 days after conception. Fortification can be an effective NTD prevention strategy in populations with limited access to folic acid foods and/or supplements. This review describes the status of mandatory FA fortification among countries that fortify (n = 71) and the research describing the impact of those programs on NTD rates (up to 78% reduction), blood folate concentrations [red blood cell folate concentrations increased ∼1.47-fold (95% CI, 1.27, 1.70) following fortification], and other health outcomes. Across settings, high-quality studies such as those with randomized exposures (e.g., randomized controlled trials, Mendelian randomization studies) are needed to elucidate interactions of FA with vitamin B12 as well as expanded biomarker testing.

Iron status and inflammation in women of reproductive age: A population-based biomarker survey and clinical study.

BACKGROUND: Women of reproductive age (WRA) are at increased risk for anemia and iron deficiency. However, there is limited population-level data in India, which could help inform evidence-based recommendations and policy. AIMS: To conduct a population-based biomarker survey of anemia, iron deficiency, and inflammation in WRA in Southern India. METHODS: Participants were WRA (15-40 y) who were not pregnant or lactating. Blood samples (n = 979) were collected and analyzed for hemoglobin (Hb), serum ferritin (SF), soluble transferrin receptor (sTfR), C-reactive protein (CRP), and alpha-1 acid glycoprotein (AGP). Anemia and severe anemia were defined as Hb < 12.0 and < 8.0 g/dL. Serum ferritin was adjusted for inflammation using BRINDA methods. Iron deficiency was defined as SF <15.0 µg/L, iron insufficiency was defined as SF < 20.0 and < 25.0 µg/L, and iron deficiency anemia was defined as Hb < 12.0 g/dL and SF < 15.0 µg/L. Inflammation was defined as CRP > 5.0 mg/L or AGP > 1.0 g/L. Restricted cubic spline regression models were also used to determine if alternative SF thresholds should be used t to classify iron deficiency. RESULTS: A total of 41.5% of WRA had anemia, and 3.0% had severe anemia. Findings from spline analyses suggested a SF cut-off of < 15.0 µg/L, consistent with conventional cut-offs for iron deficiency. 46.3% of WRA had SF < 15.0 µg/L (BRINDA-adjusted: 61.5%), 55.0% had SF < 20.0 µg/L (72.7%), 61.8% had SF < 25.0 µg/L (81.0%), and 30.0% had IDA (34.5%). 17.3% of WRA had CRP > 5.0 mg/L and 22.2% had AGP > 1.0 g/L. The prevalence of ID (rural vs. urban: 49.1% vs. 34.9%; p = 0.0004), iron insufficiency (57.8% vs. 43.8%; p = 0.0005), and IDA (31.8% vs. 22.4%; p = 0.01) were significantly higher in rural areas, although CRP levels were lower and there were no differences in elevated CRP or AGP. CONCLUSIONS: The burden of anemia and iron deficiency in this population was substantial, and increased after adjusting for inflammation, suggesting potential to benefit from screening and interventions. REGISTRATION NUMBER: NCT04048330.

Demographic, Physiologic, and Lifestyle Characteristics Observed with Serum Total Folate Differ Among Folate Forms: Cross-Sectional Data from Fasting Samples in the NHANES 2011-2016.

BACKGROUND: Serum folate forms were measured in the US population during recent NHANES to assess folate status. OBJECTIVE: We describe post-folic acid-fortification concentrations of serum folate forms in the fasting US population ≥1 y from the NHANES 2011-2016. METHODS: We measured 5 biologically active folates and 1 oxidation product (MeFox) of 5-methyltetrahydrofolate (5-methyl-THF). We calculated geometric means of 5-methyl-THF, unmetabolized folic acid (UMFA), nonmethyl folate (sum of tetrahydrofolate, 5-formyltetrahydrofolate, and 5,10-methenyltetrahydrofolate), total folate (sum of above biomarkers), and MeFox by demographic, physiologic, and lifestyle variables; estimated the magnitude of variables on biomarker concentrations after covariate adjustment; and determined the prevalence of UMFA >2 nmol/L. RESULTS: After demographic adjustment, age, sex, and race-Hispanic origin were significantly associated with most folate forms. MeFox increased with age, while 5-methyl-THF, UMFA, and nonmethyl folate displayed U-shaped age patterns. Compared with non-Hispanic whites, non-Hispanic blacks had 23% lower predicted 5-methyl-THF but comparable UMFA; non-Hispanic Asians had comparable 5-methyl-THF but 28% lower UMFA; Hispanics, non-Hispanic Asians, and non-Hispanic blacks had ∼20% lower MeFox. After additional physiologic and lifestyle adjustment, predicted UMFA and MeFox concentrations were 43% and 112% higher, respectively, in adults with chronic kidney disease and 17% and 15% lower, respectively, in adults consuming daily 1-<2 alcoholic beverages; 5-methyl-THF concentrations were 20% lower in adult smokers. The prevalence of UMFA >2 nmol/L was highest in persons aged ≥70 y (9.01%) and lowest in those aged 12-19 y (1.14%). During 2011-2014, the prevalence was 10.6% in users and 2.22% in nonusers of folic acid-containing supplements. CONCLUSIONS: In fasting persons ≥1 y, the demographic, physiologic, and lifestyle characteristics observed with serum total folate differed among folate forms, suggesting biological and/or genetic influences on folate metabolism. High UMFA was mostly observed in supplement users and older persons.

Defining the plasma folate concentration associated with the red blood cell folate concentration threshold for optimal neural tube defects prevention: a population-based, randomized trial of folic acid supplementation.

BACKGROUND: For women of reproductive age, a population-level red blood cell (RBC) folate concentration below the threshold 906 nmol/L or 400 ng/mL indicates folate insufficiency and suboptimal neural tube defect (NTD) prevention. A corresponding population plasma/serum folate concentration threshold for optimal NTD prevention has not been established. OBJECTIVE: The aim of this study was to examine the association between plasma and RBC folate concentrations and estimated a population plasma folate insufficiency threshold (pf-IT) corresponding to the RBC folate insufficiency threshold (RBCf-IT) of 906 nmol/L. METHODS: We analyzed data on women of reproductive age (n = 1673) who participated in a population-based, randomized folic acid supplementation trial in northern China. Of these women, 565 women with anemia and/or vitamin B-12 deficiency were ineligible for folic acid intervention (nonintervention group); the other 1108 received folic acid supplementation for 6 mo (intervention group). We developed a Bayesian linear model to estimate the pf-IT corresponding to RBCf-IT by time from supplementation initiation, folic acid dosage, methyltetrahydrofolate reductase (MTHFR) genotype, body mass index (BMI), vitamin B-12 status, or anemia status. RESULTS: Using plasma and RBC folate concentrations of the intervention group, the estimated median pf-IT was 25.5 nmol/L (95% credible interval: 24.6, 26.4). The median pf-ITs were similar between the baseline and postsupplementation samples (25.7 compared with 25.2 nmol/L) but differed moderately (±3-4 nmol/L) by MTHFR genotype and BMI. Using the full population-based baseline sample (intervention and nonintervention), the median pf-IT was higher for women with vitamin B-12 deficiency (34.6 nmol/L) and marginal deficiency (29.8 nmol/L) compared with the sufficient group (25.6 nmol/L). CONCLUSIONS: The relation between RBC and plasma folate concentrations was modified by BMI and genotype and substantially by low plasma vitamin B-12. This suggests that the threshold of 25.5 nmol/L for optimal NTD prevention may be appropriate in populations with similar characteristics, but it should not be used in vitamin B-12 insufficient populations. This trial was registered at clinicaltrials.gov as NCT00207558.

Modeling the impact of folic acid fortification and supplementation on red blood cell folate concentrations and predicted neural tube defect risk in the United States: have we reached optimal prevention?

Background: The US CDC and the Institute of Medicine recommend that women capable of becoming pregnant consume ≥400 µg synthetic folic acid/d to prevent neural tube defects (NTDs). The United States has 3 sources of folic acid: fortified enriched cereal grain products (ECGPs), fortified ready-to-eat (RTE) cereals, and dietary supplements. Objective: Our objectives were as follows: 1) to estimate the usual daily folic acid intake and distributions of red blood cell (RBC) folate concentrations among women consuming folic acid from different sources; 2) to assess the usual daily total folic acid intake associated with optimal RBC folate concentrations for NTD prevention; 3) to predict NTD prevalence; and 4) to estimate the number of preventable folate-sensitive NTDs. Design: NHANES data (2007-2012) for nonpregnant women of reproductive age (12-49 y) were used to estimate usual daily intakes of synthetic folic acid and natural food folate. We applied existing models of the relation between RBC folate concentrations and NTD risk to predict NTD prevalence. Results: Based on the distribution of overall RBC folate concentrations (4783 women), the predicted NTD prevalence was 7.3/10,000 live births [95% uncertainty interval (UI): 5.5-9.4/10,000 live births]. Women consuming folic acid from ECGPs as their only source had lower usual daily total folic acid intakes (median: 115 µg/d; IQR: 79-156 µg/d), lower RBC folate concentrations (median: 881 nmol/L; IQR: 704-1108 nmol/L), and higher predicted NTD prevalence (8.5/10,000 live births; 95% UI: 6.4-10.8/10,000 live births) compared with women consuming additional folic acid from diet or supplements. If women who currently consume folic acid from ECGPs only (48% of women) consumed additional folic acid sources, 345 (95% UI: 0-821) to 701 (95% UI: 242-1189) additional NTDs/y could be prevented. Conclusions: This analysis supports current recommendations and does not indicate any need for higher intakes of folic acid to achieve optimal NTD prevention. Ensuring 400 µg/d intake of folic acid prior to pregnancy has the potential to increase the number of babies born without an NTD.

Assessing the association between natural food folate intake and blood folate concentrations: a systematic review and Bayesian meta-analysis of trials and observational studies.

Folate is found naturally in foods or as synthetic folic acid in dietary supplements and fortified foods. Adequate periconceptional folic acid intake can prevent neural tube defects. Folate intake impacts blood folate concentration; however, the dose-response between natural food folate and blood folate concentrations has not been well described. We estimated this association among healthy females. A systematic literature review identified studies (1 1992-3 2014) with both natural food folate intake alone and blood folate concentration among females aged 12-49 years. Bayesian methods were used to estimate regression model parameters describing the association between natural food folate intake and subsequent blood folate concentration. Seven controlled trials and 29 observational studies met the inclusion criteria. For the six studies using microbiologic assay (MA) included in the meta-analysis, we estimate that a 6% (95% Credible Interval (CrI): 4%, 9%) increase in red blood cell (RBC) folate concentration and a 7% (95% CrI: 1%, 12%) increase in serum/plasma folate concentration can occur for every 10% increase in natural food folate intake. Using modeled results, we estimate that a natural food folate intake of ≥ 450 µg dietary folate equivalents (DFE)/day could achieve the lower bound of an RBC folate concentration (~ 1050 nmol/L) associated with the lowest risk of a neural tube defect. Natural food folate intake affects blood folate concentration and adequate intakes could help women achieve a RBC folate concentration associated with a risk of 6 neural tube defects/10,000 live births.

U.S. women of childbearing age who are at possible increased risk of a neural tube defect-affected pregnancy due to suboptimal red blood cell folate concentrations, National Health and Nutrition Examination Survey 2007 to 2012.

BACKGROUND: Red blood cell (RBC) folate concentrations are a potential biomarker of folate-sensitive neural tube defect (NTD) risk in the population. The purpose of this analysis was to describe women in the U.S. population with RBC folate concentrations below those associated with optimal NTD prevention. METHODS: We used data from the 2007 to 2012 National Health and Nutrition Examination Survey (NHANES) to assess the RBC folate status of U.S. women of childbearing age relative to risk categories for NTD risk based on RBC folate concentrations. We defined suboptimal RBC folate concentrations as those associated with a prevalence of ≥9 NTDs per 10,000 live births. RESULTS: Among nonpregnant women age 12 to 49 years, 22.8% (95% Confidence Interval: 21.1, 24.6) had suboptimal RBC folate concentrations. Women had greater odds of having a suboptimal RBC folate concentration if they did not use dietary supplements containing folic acid; had mandatorily fortified enriched cereal grain products as their only source of folic acid; were non-Hispanic black or Hispanic; or were current smokers. CONCLUSION: Based on RBC folate concentrations, we would predict that the majority of U.S. women of reproductive age are not at increased risk for folate sensitive NTDs in the presence of mandatory folic acid fortification. Prevention policies and programs can be aimed at population subgroups identified as having higher predicted risk for folate-sensitive NTDs based on RBC folate concentrations.

Assessing the association between the methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism and blood folate concentrations: a systematic review and meta-analysis of trials and observational studies.

BACKGROUND: The methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism is a risk factor for neural tube defects. The T allele produces an enzyme with reduced folate-processing capacity, which has been associated with lower blood folate concentrations. OBJECTIVE: We assessed the association between MTHFR C677T genotypes and blood folate concentrations among healthy women aged 12-49 y. DESIGN: We conducted a systematic review of the literature published from January 1992 to March 2014 to identify trials and observational studies that reported serum, plasma, or red blood cell (RBC) folate concentrations and MTHFR C677T genotype. We conducted a meta-analysis for estimates of percentage differences in blood folate concentrations between genotypes. RESULTS: Forty studies met the inclusion criteria. Of the 6 studies that used the microbiologic assay (MA) to measure serum or plasma (S/P) and RBC folate concentrations, the percentage difference between genotypes showed a clear pattern of CC > CT > TT. The percentage difference was greatest for CC > TT [S/P: 13%; 95% credible interval (CrI): 7%, 18%; RBC: 16%; 95% CrI: 12%, 20%] followed by CC > CT (S/P: 7%; 95% CrI: 1%, 12%; RBC: 8%; 95% CrI: 4%, 12%) and CT > TT (S/P: 6%; 95% CrI: 1%, 11%; RBC: 9%; 95% CrI: 5%, 13%). S/P folate concentrations measured by using protein-binding assays (PBAs) also showed this pattern but to a greater extent (e.g., CC > TT: 20%; 95% CrI: 17%, 22%). In contrast, RBC folate concentrations measured by using PBAs did not show the same pattern and are presented in the Supplemental Material only. CONCLUSIONS: Meta-analysis results (limited to the MA, the recommended population assessment method) indicated a consistent percentage difference in S/P and RBC folate concentrations across MTHFR C677T genotypes. Lower blood folate concentrations associated with this polymorphism could have implications for a population-level risk of neural tube defects.

Neural tube defects and maternal intake of micronutrients related to one-carbon metabolism or antioxidant activity.

BACKGROUND: Maternal nutritional status has been evaluated to clarify its role in development of neural tube defects (NTDs). Maternal folate intake during pregnancy has been closely evaluated for its association with NTDs. The study objective was to examine associations between NTDs and other dietary periconceptional micronutrient intake, particularly nutrients involved in one-carbon metabolism or antioxidant activity. METHODS: Using data from the National Birth Defects Prevention Study, 1997-2005, logistic regression models were used to estimate the relative risk of NTDs based on maternal micronutrient intake. RESULTS: Results were stratified according to folic acid supplement use, race/ethnicity, and maternal body mass index. Analyses included 954 cases (300 with anencephaly, 654 with spina bifida) and 6268 controls. Higher intakes of folate, thiamin, betaine, iron, and vitamin A were associated with decreased risk of anencephaly among some ethnic and clinical groups. In some groups, higher intakes of thiamin, riboflavin, vitamin B(6) , vitamin C, vitamin E, niacin, and retinol were associated with decreased risk of spina bifida. CONCLUSION: In addition to folic acid, other micronutrients, including thiamin, betaine, riboflavin, vitamin B(6) , vitamin C, vitamin E, niacin, iron, retinol, and vitamin A, may decrease the risk of NTD occurrence. Birth Defects Research (Part A) 2012. © 2012 Wiley Periodicals, Inc.

Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis.

Hedgehog signalling--an essential pathway during embryonic pancreatic development, the misregulation of which has been implicated in several forms of cancer--may also be an important mediator in human pancreatic carcinoma. Here we report that sonic hedgehog, a secreted hedgehog ligand, is abnormally expressed in pancreatic adenocarcinoma and its precursor lesions: pancreatic intraepithelial neoplasia (PanIN). Pancreata of Pdx-Shh mice (in which Shh is misexpressed in the pancreatic endoderm) develop abnormal tubular structures, a phenocopy of human PanIN-1 and -2. Moreover, these PanIN-like lesions also contain mutations in K-ras and overexpress HER-2/neu, which are genetic mutations found early in the progression of human pancreatic cancer. Furthermore, hedgehog signalling remains active in cell lines established from primary and metastatic pancreatic adenocarcinomas. Notably, inhibition of hedgehog signalling by cyclopamine induced apoptosis and blocked proliferation in a subset of the pancreatic cancer cell lines both in vitro and in vivo. These data suggest that this pathway may have an early and critical role in the genesis of this cancer, and that maintenance of hedgehog signalling is important for aberrant proliferation and tumorigenesis.