Familiar Disposition of May-Thurner Syndrome-A Case Series.

May-Thurner syndrome is a venous compression syndrome of the pelvic vessels that represents a relevant risk factor for thrombus formation. The standard procedure to secure a diagnosis is venography, followed by endovascular therapy as the preferred treatment choice if the patient is symptomatic. In our case series, there are three related patients with May-Thurner syndrome. A 16-year-old female was admitted with pulmonary embolism, dyspnoea and hip pain. The compression syndrome was diagnosed with interventional venography, and the patient received venous stent implantation. Due to her family history, we also suspected her mother to be affected by the syndrome and elucidated the diagnosis shortly afterwards by invasive venography. Subsequently, we examined the patient's 19-year-old brother, and magnetic resonance imaging confirmed May-Thurner syndrome. A similar case series has not been published before. In this case, the family relation indicates a possible hereditary aspect of May-Thurner syndrome. This hypothesis should be the subject of further research. In conclusion, it is essential to assess family history thoroughly when treating patients with May-Thurner syndrome.

Identification of de novo variants in nonsyndromic cleft lip with/without cleft palate patients with low polygenic risk scores.

BACKGROUND: Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a congenital malformation of multifactorial etiology. Research has identified >40 genome-wide significant risk loci, which explain less than 40% of nsCL/P heritability. Studies show that some of the hidden heritability is explained by rare penetrant variants. METHODS: To identify new candidate genes, we searched for highly penetrant de novo variants (DNVs) in 50 nsCL/P patient/parent-trios with a low polygenic risk for the phenotype (discovery). We prioritized DNV-carrying candidate genes from the discovery for resequencing in independent cohorts of 1010 nsCL/P patients of diverse ethnicities and 1574 population-matched controls (replication). Segregation analyses and rare variant association in the replication cohort, in combination with additional data (genome-wide association data, expression, protein-protein-interactions), were used for final prioritization. CONCLUSION: In the discovery step, 60 DNVs were identified in 60 genes, including a variant in the established nsCL/P risk gene CDH1. Re-sequencing of 32 prioritized genes led to the identification of 373 rare, likely pathogenic variants. Finally, MDN1 and PAXIP1 were prioritized as top candidates. Our findings demonstrate that DNV detection, including polygenic risk score analysis, is a powerful tool for identifying nsCL/P candidate genes, which can also be applied to other multifactorial congenital malformations.

Bitterless guaifenesin prodrugs-design, synthesis, characterization, in vitro kinetics, and bitterness studies.

A respected number of drugs suffer from bitter taste which results in patient incompliance. With the aim of solving the bitterness of guaifenesin, dimethyl maleate, maleate, glutarate, succinate, and dimethyl succinate prodrugs were designed and synthesized. Molecular orbital methods were utilized for the design of the ester prodrugs. The density functional theory (DFT) calculations revealed that the hydrolysis efficiency of the synthesized prodrugs is significantly sensitive to the pattern of substitution on C=C bond and distance between the nucleophile and the electrophile. The hydrolysis of the prodrugs was largely affected by the pH of the medium. The experimental t1/2 for the hydrolysis of guaifenesin dimaleate ester prodrugs in 1N HCl was the least and for guaifenesin dimethyl succinate was the highest. Functional heterologous expression of TAS2R14, a broadly tuned bitter taste receptor responding to guaifenesin, and experiments using these prodrugs revealed that, while some of the prodrugs still activated the receptor similarly or even stronger than the parent substance, succinate derivatization resulted in the complete loss of receptor responses. The predicted binding modes of guaifenesin and its prodrugs to the TAS2R14 homology model suggest that the decreased activity of the succinate derivatives may be caused by a clash with Phe247.

Reengineering the ligand sensitivity of the broadly tuned human bitter taste receptor TAS2R14.

BACKGROUND: In humans, bitterness perception is mediated by ~25 bitter taste receptors present in the oral cavity. Among these receptors three, TAS2R10, TAS2R14 and TAS2R46, exhibit extraordinary wide agonist profiles and hence contribute disproportionally high to the perception of bitterness. Perhaps the most broadly tuned receptor is the TAS2R14, which may represent, because of its prominent expression in extraoral tissues, a receptor of particular importance for the physiological actions of bitter compounds beyond taste. METHODS: To investigate how the architecture and composition of the TAS2R14 binding pocket enables specific interactions with a complex array of chemically diverse bitter agonists, we carried out homology modeling and ligand docking experiments, subjected the receptor to point-mutagenesis of binding site residues and performed functional calcium mobilization assays. RESULTS: In total, 40 point-mutated receptor constructs were generated to investigate the contribution of 19 positions presumably located in the receptor's binding pocket to activation by 7 different TAS2R14 agonists. All investigated positions exhibited moderate to pronounced agonist selectivity. CONCLUSIONS: Since numerous modifications of the TAS2R14 binding pocket resulted in improved responses to individual agonists, we conclude that this bitter taste receptor might represent a suitable template for the engineering of the agonist profile of a chemoreceptive receptor. GENERAL SIGNIFICANCE: The detailed structure-function analysis of the highly promiscuous and widely expressed TAS2R14 suggests that this receptor must be considered as potentially frequent target for known and novel drugs including undesired off-effects.

Nonsyndromic cleft palate: An association study at GWAS candidate loci in a multiethnic sample.

BACKGROUND: Nonsyndromic cleft palate only (nsCPO) is a common and multifactorial form of orofacial clefting. In contrast to successes achieved for the other common form of orofacial clefting, that is, nonsyndromic cleft lip with/without cleft palate (nsCL/P), genome wide association studies (GWAS) of nsCPO have identified only one genome wide significant locus. Aim of the present study was to investigate whether common variants contribute to nsCPO and, if so, to identify novel risk loci. METHODS: We genotyped 33 SNPs at 27 candidate loci from 2 previously published nsCPO GWAS in an independent multiethnic sample. It included: (i) a family-based sample of European ancestry (n = 212); and (ii) two case/control samples of Central European (n = 94/339) and Arabian ancestry (n = 38/231), respectively. A separate association analysis was performed for each genotyped dataset, and meta-analyses were performed. RESULTS: After association analysis and meta-analyses, none of the 33 SNPs showed genome-wide significance. Two variants showed nominally significant association in the imputed GWAS dataset and exhibited a further decrease in p-value in a European and an overall meta-analysis including imputed GWAS data, respectively (rs395572: PMetaEU = 3.16 × 10-4 ; rs6809420: PMetaAll = 2.80 × 10-4 ). CONCLUSION: Our findings suggest that there is a limited contribution of common variants to nsCPO. However, the individual effect sizes might be too small for detection of further associations in the present sample sizes. Rare variants may play a more substantial role in nsCPO than in nsCL/P, for which GWAS of smaller sample sizes have identified genome-wide significant loci. Whole-exome/genome sequencing studies of nsCPO are now warranted.

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.

Probing the Binding Pocket of the Broadly Tuned Human Bitter Taste Receptor TAS2R14 by Chemical Modification of Cognate Agonists.

Sensing potentially harmful bitter substances in the oral cavity is achieved by a group of (˜) 25 receptors, named TAS2Rs, which are expressed in specialized sensory cells and recognize individual but overlapping sets of bitter compounds. The receptors differ in their tuning breadths ranging from narrowly to broadly tuned receptors. One of the most broadly tuned human bitter taste receptors is the TAS2R14 recognizing an enormous variety of chemically diverse synthetic and natural bitter compounds, including numerous medicinal drugs. This suggests that this receptor possesses a large readily accessible ligand binding pocket. To allow probing the accessibility and size of the ligand binding pocket, we chemically modified cognate agonists and tested receptor responses in functional assays. The addition of large functional groups to agonists was usually possible without abolishing agonistic activity. The newly synthesized agonist derivatives were modeled in the binding site of the receptor, providing comparison to the mother substances and rationalization of the in vitro activities of this series of compounds.

Evaluating eight newly identified susceptibility loci for nonsyndromic cleft lip with or without cleft palate in a Mesoamerican population.

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is among the most frequently occurring congenital malformations worldwide. The number of genetic loci identified as being involved in NSCL/P etiology was recently increased by a large genome-wide meta-analysis of European and Asian samples. This meta-analysis confirmed all six previously recognized genetic susceptibility loci and identified six novel ones. METHODS: To investigate which of these 12 loci contribute to NSCL/P risk in an independent sample of distinct ethnicity, we performed a case-control association analysis in a sample of the Mesoamerican population. A total of 153 individuals with NSCL/P (cases) and 337 unaffected controls were included. Top single-nucleotide polymorphisms (SNPs) at 8 of the 12 loci (1p22.1, 1p36, 2p21, 3p11.1, 8q21.3, 13q31.1, 15q22, and 20q12) were analyzed using mass spectroscopy and restriction-length-fragment polymorphism analyses. In a previous study, we had analyzed the remaining four NSCL/P susceptibility regions (IRF6, 8q24, 10q25, and 17q22) in the same sample. RESULTS: Single-marker association analyses applying allelic, dominant, and recessive models revealed nominal significant associations for four of the eight loci, with two additional loci showing at least a trend of association in the hypothesized direction. CONCLUSION: In combination with results from our previous study using the same sample, our data suggest that the majority of the known NSCL/P susceptibility regions identified to date also confer risk for this malformation in the Mesoamerican population. Birth Defects Research (Part A) 100:43-47, 2014. © 2013 Wiley Periodicals, Inc.

The bitter pill: clinical drugs that activate the human bitter taste receptor TAS2R14.

Bitter taste receptors (TAS2Rs) mediate aversive response to toxic food, which is often bitter. These G-protein-coupled receptors are also expressed in extraoral tissues, and emerge as novel targets for therapeutic indications such as asthma and infection. Our goal was to identify ligands of the broadly tuned TAS2R14 among clinical drugs. Molecular properties of known human bitter taste receptor TAS2R14 agonists were incorporated into pharmacophore- and shape-based models and used to computationally predict additional ligands. Predictions were tested by calcium imaging of TAS2R14-transfected HEK293 cells. In vitro testing of the virtual screening predictions resulted in 30-80% success rates, and 15 clinical drugs were found to activate the TAS2R14. hERG potassium channel, which is predominantly expressed in the heart, emerged as a common off-target of bitter drugs. Despite immense chemical diversity of known TAS2R14 ligands, novel ligands and previously unknown polypharmacology of drugs were unraveled by in vitro screening of computational predictions. This enables rational repurposing of traditional and standard drugs for bitter taste signaling modulation for therapeutic indications.

Resequencing of VAX1 in patients with nonsyndromic cleft lip with or without cleft palate.

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common of all congenital anomalies, and has a multifactorial etiology involving both environmental and genetic factors. Recent genome-wide association studies (GWAS) identified strong association between a locus on chromosome 10q25.3 and NSCL/P in European samples. One gene at 10q25.3, the ventral anterior homeobox 1 (VAX1) gene, is considered a strong candidate gene for craniofacial malformations. The purpose of the present study was to provide further evidence that VAX1 is the causal gene at the 10q25.3 locus through identification of an excess of rare mutations in patients with NSCL/P. METHODS: The 5'UTR, complete coding regions, and adjacent splice sites of the two known VAX1 isoforms were sequenced in 384 patients with NSCL/P and 384 controls of Central European descent. Observed variants were investigated with respect to familial cosegregation or de novo occurrence, and in silico analyses were performed to identify putative effects on the transcript or protein level. RESULTS: Eighteen single-base variants were found, 15 of them rare and previously unreported. In the long VAX1 isoform, predicted functionally relevant variants were observed more often in NSCL/P cases, although this difference was not significant (p = 0.17). Analysis of family members demonstrated incomplete cosegregation in most pedigrees. CONCLUSION: Our data do not support the hypothesis that highly penetrant rare variants in VAX1 are a cause of NSCL/P. To determine whether VAX1 is the causative gene at 10q25.3 further research, in particular into the biologic function of its long isoform, is warranted. Birth Defects Research (Part A), 2012.