High amount of fertility reducing tumors and procedures, but no evidence for premature ovarian failure in female Lynch syndrome patients.

Lynch syndrome (LS; HNPCC) patients carry heterozygous pathogenic germline variants in mismatch repair (MMR) genes, which have also been shown to play an important role in meiosis. Therefore, it was hypothesized, that LS might be associated with a higher risk for premature ovarian failure (POF) or earlier menopause. Data on medical gynaecological history, cancer diagnoses and therapy were collected from 167 female LS patients and compared to a population-based control cohort. There was no difference between the age of menopause in patients compared to controls and no evidence for a higher risk of POF in LS patients. However, around one third (35%) of the probands have already had premenopausal cancer and mostly cancer-related treatment affecting fertility before the age of 45 years. Therefore, childbearing time might still be limited in these patients, especially due to the premenopausal cancer risk. LS patients should be informed in time about the elevated premenopausal cancer risks and the possible impact on family planning. This is particularly relevant since the average childbearing age has increased during the last decades.

Dynamic Domain Links Substrate Binding and Catalysis in the Factor-Inhibiting-HIF-1.

The interplay between active-site chemistry and functionally relevant enzyme motions can provide useful insights into selective enzyme modulation. Modulation of the hypoxia-sensing function of factor-inhibiting-HIF-1 (FIH) enzyme is a potential therapeutic strategy in disease states such as ischemia and cancer. The hypoxia-sensing function of FIH relies in major part on the tight coupling of the first half of the catalytic mechanism which involves O2 activation and eventual succinate production to the second half which involves HIF-1α/CTAD substrate hydroxylation. In this study, we demonstrate the role of a loop hinge domain in FIH (FIH102-118) called the 100s loop in maintaining this particular tight coupling. Molecular dynamics patterns from Gaussian Network Model (iGNM) database analysis of FIH identified the 100s loop as one dynamic domain containing a hinge residue (Tyr102) with a potential substrate positioning role. Enzymological and biophysical studies of the 100s loop point mutants revealed altered enzyme kinetics with the exception of the conservative FIH mutant Y102F, which suggests a sterics-related role for this residue. Removal of the bulk of Tyr102 (Y102A) resulted in succinate production, autohydroxylation, and an O2 binding environment comparable to wild-type FIH. However, the HIF-1α/CTAD substrate hydroxylation of this mutant was significantly reduced which implies that (1) the FIH loop hinge residue Tyr102 does not affect O2 activation, (2) the stacking steric interaction of Tyr102 is important in substrate positioning for productive hydroxylation, and (3) Tyr102 is important for the synchronization of O2 activation and substrate hydroxylation.

Dissecting the genetic heterogeneity of gastric cancer.

BACKGROUND: Gastric cancer (GC) is clinically heterogenous according to location (cardia/non-cardia) and histopathology (diffuse/intestinal). We aimed to characterize the genetic risk architecture of GC according to its subtypes. Another aim was to examine whether cardia GC and oesophageal adenocarcinoma (OAC) and its precursor lesion Barrett's oesophagus (BO), which are all located at the gastro-oesophageal junction (GOJ), share polygenic risk architecture. METHODS: We did a meta-analysis of ten European genome-wide association studies (GWAS) of GC and its subtypes. All patients had a histopathologically confirmed diagnosis of gastric adenocarcinoma. For the identification of risk genes among GWAS loci we did a transcriptome-wide association study (TWAS) and expression quantitative trait locus (eQTL) study from gastric corpus and antrum mucosa. To test whether cardia GC and OAC/BO share genetic aetiology we also used a European GWAS sample with OAC/BO. FINDINGS: Our GWAS consisting of 5816 patients and 10,999 controls highlights the genetic heterogeneity of GC according to its subtypes. We newly identified two and replicated five GC risk loci, all of them with subtype-specific association. The gastric transcriptome data consisting of 361 corpus and 342 antrum mucosa samples revealed that an upregulated expression of MUC1, ANKRD50, PTGER4, and PSCA are plausible GC-pathomechanisms at four GWAS loci. At another risk locus, we found that the blood-group 0 exerts protective effects for non-cardia and diffuse GC, while blood-group A increases risk for both GC subtypes. Furthermore, our GWAS on cardia GC and OAC/BO (10,279 patients, 16,527 controls) showed that both cancer entities share genetic aetiology at the polygenic level and identified two new risk loci on the single-marker level. INTERPRETATION: Our findings show that the pathophysiology of GC is genetically heterogenous according to location and histopathology. Moreover, our findings point to common molecular mechanisms underlying cardia GC and OAC/BO. FUNDING: German Research Foundation (DFG).

Measurement of kinetic isotope effects on peptide hydroxylation using MALDI-MS.

Primary kinetic isotope effects (KIEs) provide unique insight into enzymatic reactions, as they can reveal rate-limiting steps and detailed chemical mechanisms. HIF hydroxylases, part of a family of 2-oxoglutarate (2OG) oxygenases are central to the regulation of many crucial biological processes through O2-sensing, but present a challenge to monitor due to the large size of the protein substrate and the similarity between native and hydroxylated substrate. MALDI-TOF MS is a convenient tool to measure peptide masses, which can also be used to measure the discontinuous kinetics of peptide hydroxylation for Factor Inhibiting HIF (FIH). Using this technique, rate data can be observed from the mole-fraction of CTAD and CTAD-OH in small volumes, allowing noncompetitive H/D KIEs to be measured. Slow dCTAD substrate leads to extensive uncoupling of O2 consumption from peptide hydroxylation, leading to enzyme autohydroxylation, which is observed using UV-vis spectroscopy. Simultaneously measuring both the normal product, CTAD-OH, and the uncoupled product, autohydroxylated enzyme, the KIE on the microscopic step of hydrogen atom transfer (HAT) can be estimated. MALDI-MS analysis is a strong method for monitoring reactions that hydroxylate peptides, and can be generalized to other similar reactions, and simultaneous kinetic detection of branched products can provide valuable insight on microscopic KIEs at intermediate mechanistic steps.

eQTL Set-Based Association Analysis Identifies Novel Susceptibility Loci for Barrett Esophagus and Esophageal Adenocarcinoma.

BACKGROUND: Over 20 susceptibility single-nucleotide polymorphisms (SNP) have been identified for esophageal adenocarcinoma (EAC) and its precursor, Barrett esophagus (BE), explaining a small portion of heritability. METHODS: Using genetic data from 4,323 BE and 4,116 EAC patients aggregated by international consortia including the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON), we conducted a comprehensive transcriptome-wide association study (TWAS) for BE/EAC, leveraging Genotype Tissue Expression (GTEx) gene-expression data from six tissue types of plausible relevance to EAC etiology: mucosa and muscularis from the esophagus, gastroesophageal (GE) junction, stomach, whole blood, and visceral adipose. Two analytical approaches were taken: standard TWAS using the predicted gene expression from local expression quantitative trait loci (eQTL), and set-based SKAT association using selected eQTLs that predict the gene expression. RESULTS: Although the standard approach did not identify significant signals, the eQTL set-based approach identified eight novel associations, three of which were validated in independent external data (eQTL SNP sets for EXOC3, ZNF641, and HSP90AA1). CONCLUSIONS: This study identified novel genetic susceptibility loci for EAC and BE using an eQTL set-based genetic association approach. IMPACT: This study expanded the pool of genetic susceptibility loci for EAC and BE, suggesting the potential of the eQTL set-based genetic association approach as an alternative method for TWAS analysis.

Allele-specific transcription factor binding in a cellular model of orofacial clefting.

Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a frequent congenital malformation with multifactorial etiology. While recent genome-wide association studies (GWAS) have identified several nsCL/P risk loci, the functional effects of the associated non-coding variants are largely unknown. Furthermore, additional risk loci remain undetected due to lack of power. As genetic variants might alter binding of transcription factors (TF), we here hypothesized that the integration of data from TF binding sites, expression analyses and nsCL/P GWAS might help to (i) identify functionally relevant variants at GWAS loci, and (ii) highlight novel risk variants that have been previously undetected. Analysing the craniofacial TF TFAP2A in human embryonic palatal mesenchyme (HEPM) cells, we identified 2845 TFAP2A ChIP-seq peaks, several of which were located near nsCL/P candidate genes (e.g. MSX1 and SPRY2). Comparison with independent data suggest that 802 of them might be specific to craniofacial development, and genes near these peaks are enriched in processes relevant to nsCL/P. Integration with nsCL/P GWAS data, however, did not show robust evidence for co-localization of common nsCL/P risk variants with TFAP2A ChIP-seq peaks. This data set represents a new resource for the analyses of craniofacial processes, and similar approaches with additional cell lines and TFs could be applied to generate further insights into nsCL/P etiology.

Incidence and predictors of ophthalmic artery occlusion in intra-arterial chemotherapy for retinoblastoma.

BACKGROUND: Retinoblastoma is the most common primary intraocular malignancy in children. OBJECTIVE: To determine the incidence of ophthalmic artery (OA) occlusion in patients treated with selective ophthalmic artery catheterization (OAC) for chemotherapy infusion for retinoblastoma. Also, to evaluate technical, anatomical, tumorous, and patient-related factors that are predictors of OA occlusion. METHODS: A retrospective chart review was performed for patients diagnosed with intraocular retinoblastoma and managed with intra-arterial chemotherapy (IAC). RESULTS: The total study cohort included 208 retinoblastoma tumors of 208 eyes in 197 consecutive patients who underwent 688 attempted IAC infusions overall with a total of 624 successful OAC infusions. The total incidence of ophthalmic artery thrombosis was 11.1% (23/208). The numbers of successful OAC procedures before diagnosing OA occlusion were one OAC in six cases (27.3%), two in seven cases (31.8%), three in four cases (18.2%), four in one case (4.5%), five in two cases (9.1%), and six in one case (4.5%). CONCLUSIONS: Local factors relating to the chemotherapy and selective microcatheterization of the OA are essential factors in the development of OA thrombosis, as seen by the association of OA thrombosis with the frequency of IAC.

Germline variation in the insulin-like growth factor pathway and risk of Barrett's esophagus and esophageal adenocarcinoma.

Genome-wide association studies (GWAS) of esophageal adenocarcinoma (EAC) and its precursor, Barrett's esophagus (BE), have uncovered significant genetic components of risk, but most heritability remains unexplained. Targeted assessment of genetic variation in biologically relevant pathways using novel analytical approaches may identify missed susceptibility signals. Central obesity, a key BE/EAC risk factor, is linked to systemic inflammation, altered hormonal signaling and insulin-like growth factor (IGF) axis dysfunction. Here, we assessed IGF-related genetic variation and risk of BE and EAC. Principal component analysis was employed to evaluate pathway-level and gene-level associations with BE/EAC, using genotypes for 270 single-nucleotide polymorphisms (SNPs) in or near 12 IGF-related genes, ascertained from 3295 BE cases, 2515 EAC cases and 3207 controls in the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) GWAS. Gene-level signals were assessed using Multi-marker Analysis of GenoMic Annotation (MAGMA) and SNP summary statistics from BEACON and an expanded GWAS meta-analysis (6167 BE cases, 4112 EAC cases, 17 159 controls). Global variation in the IGF pathway was associated with risk of BE (P = 0.0015). Gene-level associations with BE were observed for GHR (growth hormone receptor; P = 0.00046, false discovery rate q = 0.0056) and IGF1R (IGF1 receptor; P = 0.0090, q = 0.0542). These gene-level signals remained significant at q < 0.1 when assessed using data from the largest available BE/EAC GWAS meta-analysis. No significant associations were observed for EAC. This study represents the most comprehensive evaluation to date of inherited genetic variation in the IGF pathway and BE/EAC risk, providing novel evidence that variation in two genes encoding cell-surface receptors, GHR and IGF1R, may influence risk of BE.

Sex-Specific Genetic Associations for Barrett's Esophagus and Esophageal Adenocarcinoma.

BACKGROUND & AIMS: Esophageal adenocarcinoma (EA) and its premalignant lesion, Barrett's esophagus (BE), are characterized by a strong and yet unexplained male predominance (with a male-to-female ratio in EA incidence of up to 6:1). Genome-wide association studies (GWAS) have identified more than 20 susceptibility loci for these conditions. However, potential sex differences in genetic associations with BE/EA remain largely unexplored. METHODS: Given strong genetic overlap, BE and EA cases were combined into a single case group for analysis. These were compared with population-based controls. We performed sex-specific GWAS of BE/EA in 3 separate studies and then used fixed-effects meta-analysis to provide summary estimates for >9 million variants for male and female individuals. A series of downstream analyses were conducted separately in male and female individuals to identify genes associated with BE/EA and the genetic correlations between BE/EA and other traits. RESULTS: We included 6758 male BE/EA cases, 7489 male controls, 1670 female BE/EA cases, and 6174 female controls. After Bonferroni correction, our meta-analysis of sex-specific GWAS identified 1 variant at chromosome 6q11.1 (rs112894788, KHDRBS2-MTRNR2L9, PBONF = .039) that was statistically significantly associated with BE/EA risk in male individuals only, and 1 variant at chromosome 8p23.1 (rs13259457, PRSS55-RP1L1, PBONF = 0.057) associated, at borderline significance, with BE/EA risk in female individuals only. We also observed strong genetic correlations of BE/EA with gastroesophageal reflux disease in male individuals and obesity in female individuals. CONCLUSIONS: The identified novel sex-specific variants associated with BE/EA could improve the understanding of the genetic architecture of the disease and the reasons for the male predominance.

Shared Genetic Etiology of Obesity-Related Traits and Barrett's Esophagus/Adenocarcinoma: Insights from Genome-Wide Association Studies.

BACKGROUND: Obesity is a major risk factor for esophageal adenocarcinoma (EA) and its precursor Barrett's esophagus (BE). Research suggests that individuals with high genetic risk to obesity have a higher BE/EA risk. To facilitate understanding of biological factors that lead to progression from BE to EA, the present study investigated the shared genetic background of BE/EA and obesity-related traits. METHODS: Cross-trait linkage disequilibrium score regression was applied to summary statistics from genome-wide association meta-analyses on BE/EA and on obesity traits. Body mass index (BMI) was used as a proxy for general obesity, and waist-to-hip ratio (WHR) for abdominal obesity. For single marker analyses, all genome-wide significant risk alleles for BMI and WHR were compared with summary statistics of the BE/EA meta-analyses. RESULTS: Sex-combined analyses revealed a significant genetic correlation between BMI and BE/EA (rg = 0.13, P = 2 × 10-04) and a rg of 0.12 between WHR and BE/EA (P = 1 × 10-02). Sex-specific analyses revealed a pronounced genetic correlation between BMI and EA in females (rg = 0.17, P = 1.2 × 10-03), and WHR and EA in males (rg = 0.18, P = 1.51 × 10-02). On the single marker level, significant enrichment of concordant effects was observed for BMI and BE/EA risk variants (P = 8.45 × 10-03) and WHR and BE/EA risk variants (P = 2 × 10-02). CONCLUSIONS: Our study provides evidence for sex-specific genetic correlations that might reflect specific biological mecha-nisms. The data demonstrate that shared genetic factors are particularly relevant in progression from BE to EA. IMPACT: Our study quantifies the genetic correlation between BE/EA and obesity. Further research is now warranted to elucidate these effects and to understand the shared pathophysiology.

p63 establishes epithelial enhancers at critical craniofacial development genes.

The transcription factor p63 is a key mediator of epidermal development. Point mutations in p63 in patients lead to developmental defects, including orofacial clefting. To date, knowledge on how pivotal the role of p63 is in human craniofacial development is limited. Using an inducible transdifferentiation model, combined with epigenomic sequencing and multicohort meta-analysis of genome-wide association studies data, we show that p63 establishes enhancers at craniofacial development genes to modulate their transcription. Disease-specific substitution mutation in the DNA binding domain or sterile alpha motif protein interaction domain of p63, respectively, eliminates or reduces establishment of these enhancers. We show that enhancers established by p63 are highly enriched for single-nucleotide polymorphisms associated with nonsyndromic cleft lip ± cleft palate (CL/P). These orthogonal approaches indicate a strong molecular link between p63 enhancer function and CL/P, illuminating molecular mechanisms underlying this developmental defect and revealing vital regulatory elements and new candidate causative genes.

No Association Between Vitamin D Status and Risk of Barrett's Esophagus or Esophageal Adenocarcinoma: A Mendelian Randomization Study.

BACKGROUND & AIMS: Epidemiology studies of circulating concentrations of 25 hydroxy vitamin D (25(OH)D) and risk of esophageal adenocarcinoma (EAC) have produced conflicting results. We conducted a Mendelian randomization study to determine the associations between circulating concentrations of 25(OH)D and risks of EAC and its precursor, Barrett's esophagus (BE). METHODS: We conducted a Mendelian randomization study using a 2-sample (summary data) approach. Six single-nucleotide polymorphisms (SNPs; rs3755967, rs10741657, rs12785878, rs10745742, rs8018720, and rs17216707) associated with circulating concentrations of 25(OH)D were used as instrumental variables. We collected data from 6167 patients with BE, 4112 patients with EAC, and 17,159 individuals without BE or EAC (controls) participating in the Barrett's and Esophageal Adenocarcinoma Consortium, as well as studies from Bonn, Germany, and Cambridge and Oxford, United Kingdom. Analyses were performed separately for BE and EAC. RESULTS: Overall, we found no evidence for an association between genetically estimated 25(OH)D concentration and risk of BE or EAC. The odds ratio per 20 nmol/L increase in genetically estimated 25(OH)D concentration for BE risk estimated by combining the individual SNP association using inverse variance weighting was 1.21 (95% CI, 0.77-1.92; P = .41). The odds ratio for EAC risk, estimated by combining the individual SNP association using inverse variance weighting, was 0.68 (95% CI, 0.39-1.19; P = .18). CONCLUSIONS: In a Mendelian randomization study, we found that low genetically estimated 25(OH)D concentrations were not associated with risk of BE or EAC.

Identification of loci of functional relevance to Barrett's esophagus and esophageal adenocarcinoma: Cross-referencing of expression quantitative trait loci data from disease-relevant tissues with genetic association data.

Esophageal adenocarcinoma (EA) and its precancerous condition Barrett's esophagus (BE) are multifactorial diseases with rising prevalence rates in Western populations. A recent meta-analysis of genome-wide association studies (GWAS) data identified 14 BE/EA risk loci located in non-coding genomic regions. Knowledge about the impact of non-coding variation on disease pathology is incomplete and needs further investigation. The aim of the present study was (i) to identify candidate genes of functional relevance to BE/EA at known risk loci and (ii) to find novel risk loci among the suggestively associated variants through the integration of expression quantitative trait loci (eQTL) and genetic association data. eQTL data from two BE/EA-relevant tissues (esophageal mucosa and gastroesophageal junction) generated within the context of the GTEx project were cross-referenced with the GWAS meta-analysis data. Variants representing an eQTL in at least one of the two tissues were categorized into genome-wide significant loci (P < 5×10-8) and novel candidate loci (5×10-8 ≤ P ≤ 5×10-5). To follow up these novel candidate loci, a genetic association study was performed in a replication cohort comprising 1,993 cases and 967 controls followed by a combined analysis with the GWAS meta-analysis data. The cross-referencing of eQTL and genetic data yielded 2,180 variants that represented 25 loci. Among the previously reported genome-wide significant loci, 22 eQTLs were identified in esophageal mucosa and/or gastroesophageal junction tissue. The regulated genes, most of which have not been linked to BE/EA etiology so far, included C2orf43/LDAH, ZFP57, and SLC9A3. Among the novel candidate loci, replication was achieved for two variants (rs7754014, Pcombined = 3.16×10-7 and rs1540, Pcombined = 4.16×10-6) which represent eQTLs for CFDP1 and SLC22A3, respectively. In summary, the present approach identified candidate genes whose expression was regulated by risk variants in disease-relevant tissues. These findings may facilitate the elucidation of BE/EA pathophysiology.

Chloride Supports O2 Activation in the D201G Facial Triad Variant of Factor-Inhibiting Hypoxia Inducible Factor, an α-Ketoglutarate Dependent Oxygenase.

α-Ketoglutarate (αKG) dependent oxygenases comprise a large superfamily of enzymes that activate O2 for varied reactions. While most of these enzymes contain a nonheme Fe bound by a His2(Asp/Glu) facial triad, a small number of αKG-dependent halogenases require only the two His ligands to bind Fe and activate O2. The enzyme "factor inhibiting HIF" (FIH) contains a His2Asp facial triad and selectively hydroxylates polypeptides; however, removal of the Asp ligand in the Asp201→Gly variant leads to a highly active enzyme, seemingly without a complete facial triad. Herein, we report on the formation of an Fe-Cl cofactor structure for the Asp201→Gly FIH variant using X-ray absorption spectroscopy (XAS), which provides insight into the structure of the His2Cl facial triad found in halogenases. The Asp201→Gly variant supports anion dependent peptide hydroxylation, demonstrating the requirement for a complete His2X facial triad to support O2 reactivity. Our results indicated that exogenous ligand binding to form a complete His2X facial triad was essential for O2 activation and provides a structural model for the His2Cl-bound nonheme Fe found in halogenases.

On engagement with anthropology: A critical evaluation of skeletal and developmental abnormalities in the Atacama preterm baby and issues of forensic and bioarchaeological research ethics. Response to Bhattacharya et al. "Whole-genome sequencing of Atacama skeleton shows novel mutations linked with dysplasia" in Genome Research, 2018, 28: 423-431. Doi: 10.1101/gr.223693.117.

Here we evaluate Bhattacharya et al.'s (2018) recent paper "Whole-genome sequencing of Atacama skeleton shows novel mutations linked with dysplasia" published in Genome Research. In this short report, we examine the hypothesis that the so-called "Atacama skeleton" has skeletal abnormalities indicative of dysplasia, critique the validity of the interpretations of disease based on genomic analyses, and comment on the ethics of research on this partially mummified human foetus. The current paper acts as a case study of the importance of using an anthropological approach for aDNA research on human remains. A critical evaluation of the ethically controversial paper by Bhattacharya et al. highlights how an understanding of skeletal biological processes, including normal and abnormal growth and development, taphonomic processes, environmental context, and close attention to ethical issues of dealing with human remains, is vital to scientific interpretations. To this end, close collaboration with palaeopathologists and local archaeologists through appropriate peer-reviewed journals will add to the rigour of scientific interpretation and circumvent misinterpretation.

Nonsyndromic cleft lip with or without cleft palate and cancer: Evaluation of a possible common genetic background through the analysis of GWAS data.

Previous research suggests a genetic overlap between nonsyndromic cleft lip with or without cleft palate (NSCL/P) and cancer. The aim of the present study was to identify common genetic risk loci for NSCL/P and cancer entities that have been reported to co-occur with orofacial clefting. This was achieved through the investigation of large genome-wide association study datasets. Investigations of 12 NSCL/P single nucleotide polymorphisms (SNPs) in 32 cancer datasets, and 204 cancer SNPs in two NSCL/P datasets, were performed. The SNPs rs13041247 (20q12) and rs6457327 (6p21.33) showed suggestive evidence for an association with both NSCL/P and a specific cancer entity. These loci harbor genes of biological relevance to oncogenesis (MAFB and OCT4, respectively). This study is the first to characterize possible pleiotropic risk loci for NSCL/P and cancer in a systematic manner. The data represent a starting point for future research by identifying a genetic link between NSCL/P and cancer.

Sequencing the GRHL3 Coding Region Reveals Rare Truncating Mutations and a Common Susceptibility Variant for Nonsyndromic Cleft Palate.

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10(-5); ORallelic = 2.46 [95% CI 1.6-3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10(-9)). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO.

The Barrett-associated variants at GDF7 and TBX5 also increase esophageal adenocarcinoma risk.

Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) represent two stages within the esophagitis-metaplasia-dysplasia-adenocarcinoma sequence. Previously genetic risk factors have been identified that confer risk to BE and EAC development. However, to which extent the genetic variants confer risk to different stages of the BE/EAC sequence remains mainly unknown. In this study we analyzed three most recently identified BE variants at the genes GDF7 (rs3072), TBX5 (rs2701108), and ALDH1A2 (rs3784262) separately in BE and EAC samples in order to determine their risk effects during BE/EAC sequence. Our data show that rs3072 at GDF7 and rs2701108 at TBX5 are also associated with EAC and conclude that both loci confer disease risk also at later stages of the BE/EAC sequence. In contrast, rs3784262 at ALDH1A2 was highly significantly associated with BE, but showed no association with EAC. Our data do not provide evidence that the ALDH1A2 locus confers equal risk in early and late stages of BE/EAC sequence.

Substrate Promotes Productive Gas Binding in the α-Ketoglutarate-Dependent Oxygenase FIH.

The Fe(2+)/α-ketoglutarate (αKG)-dependent oxygenases use molecular oxygen to conduct a wide variety of reactions with important biological implications, such as DNA base excision repair, histone demethylation, and the cellular hypoxia response. These enzymes follow a sequential mechanism in which O2 binds and reacts after the primary substrate binds, making those structural factors that promote productive O2 binding central to their chemistry. A large challenge in this field is to identify strategies that engender productive turnover. Factor inhibiting HIF (FIH) is a Fe(2+)/αKG-dependent oxygenase that forms part of the O2 sensing machinery in human cells by hydroxylating the C-terminal transactivation domain (CTAD) found within the HIF-1α protein. The structure of FIH was determined with the O2 analogue NO bound to Fe, offering the first direct insight into the gas binding geometry in this enzyme. Through a combination of density functional theory calculations, {FeNO}(7) electron paramagnetic resonance spectroscopy, and ultraviolet-visible absorption spectroscopy, we demonstrate that CTAD binding stimulates O2 reactivity by altering the orientation of the bound gas molecule. Although unliganded FIH binds NO with moderate affinity, the bound gas can adopt either of two orientations with similar stability; upon CTAD binding, NO adopts a single preferred orientation that is appropriate for supporting oxidative decarboxylation. Combined with other studies of related enzymes, our data suggest that substrate-induced reorientation of bound O2 is the mechanism utilized by the αKG oxygenases to tightly couple O2 activation to substrate hydroxylation.