Viable <Emphasis Type="Italic">Ednra</Emphasis><Superscript><Emphasis Type="Italic">Y129F</Emphasis></Superscript> mice feature human mandibulofacial dysostosis with alopecia (MFDA) syndrome due to the homologue mutation

Animal models resembling human mutations are valuable tools to research the features of complex human craniofacial syndromes. This is the first report on a viable dominant mouse model carrying a non-synonymous sequence variation within the endothelin receptor type A gene (Ednra c.386A>T, p.Tyr129Phe) derived by an ENU mutagenesis program. The identical amino acid substitution was reported recently as disease causing in three individuals with the mandibulofacial dysostosis with alopecia (MFDA, OMIM 616367) syndrome. We performed standardized phenotyping of wild-type, heterozygous, and homozygous Ednra ^Y129F mice within the German Mouse Clinic. Mutant mice mimic the craniofacial phenotypes of jaw dysplasia, micrognathia, dysplastic temporomandibular joints, auricular dysmorphism, and missing of the squamosal zygomatic process as described for MFDA-affected individuals. As observed in MFDA-affected individuals, mutant Ednra ^Y129F mice exhibit hearing impairment in line with strong abnormalities of the ossicles and further, reduction of some lung volumetric parameters. In general, heterozygous and homozygous mice demonstrated inter-individual diversity of expression of the craniofacial phenotypes as observed in MFDA patients but without showing any cleft palates, eyelid defects, or alopecia. Mutant Ednra ^Y129F mice represent a valuable viable model for complex human syndromes of the first and second pharyngeal arches and for further studies and analysis of impaired endothelin 1 (EDN1)–endothelin receptor type A (EDNRA) signaling. Above all, Ednra ^Y129F mice model the recently published human MFDA syndrome and may be helpful for further disease understanding and development of therapeutic interventions.     

Mutations in Endothelin 1 Cause Recessive Auriculocondylar Syndrome and Dominant Isolated Question-Mark Ears

Auriculocondylar syndrome (ACS) is a rare craniofacial disorder with mandibular hypoplasia and question-mark ears (QMEs) as major features. QMEs, consisting of a specific defect at the lobe-helix junction, can also occur as an isolated anomaly. Studies in animal models have indicated the essential role of endothelin 1 (EDN1) signaling through the endothelin receptor type A (EDNRA) in patterning the mandibular portion of the first pharyngeal arch. Mutations in the genes coding for phospholipase C, beta 4 (PLCB4) and guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 3 (GNAI3), predicted to function as signal transducers downstream of EDNRA, have recently been reported in ACS. By whole-exome sequencing (WES), we identified a homozygous substitution in a furin cleavage site of the EDN1 proprotein in ACS-affected siblings born to consanguineous parents. WES of two cases with vertical transmission of isolated QMEs revealed a stop mutation in EDN1 in one family and a missense substitution of a highly conserved residue in the mature EDN1 peptide in the other. Targeted sequencing of EDN1 in an ACS individual with related parents identified a fourth, homozygous mutation falling close to the site of cleavage by endothelin-converting enzyme. The different modes of inheritance suggest that the degree of residual EDN1 activity differs depending on the mutation. These findings provide further support for the hypothesis that ACS and QMEs are uniquely caused by disruption of the EDN1-EDNRA signaling pathway.     

Effects of endothelin receptor type-A and type-B antagonists on prostaglandin F2alpha-induced luteolysis of the sheep corpus luteum

Three experiments were designed to examine the mechanisms that govern prostaglandin (PGF2alpha)-induced regression of the sheep corpus luteum. Evidence is presented supporting the involvement of endothelin 1 (EDN1) in PGF2alpha-induced luteolysis. Experiment 1 measured effects of PGF2alpha when actions of EDN1 were blocked by sustained administration of a type-A endothelin (EDNRA) or type-B endothelin (EDNRB) antagonist in vivo. Experiment 2 examined antisteroidogenic actions of PGF2alpha and EDN1 in the presence of an EDNRA or EDNRB antagonist in Day-8 luteal minces. In experiment 3, luteal cellular expression of EDNRA and EDNRB was determined immunohistochemically. Relative gene expression of EDNRA and EDNRB receptors was examined by real-time RT-PCR in Day-8 sheep corpora lutea. EDNRA, but not EDNRB, participated in antisteroidogenic actions of EDN1. During the first 12 h after PGF2alpha-induced luteolysis, EDNRA antagonist did not prevent a decline in serum progesterone concentrations. Early actions of PGF2alpha are either direct or mediated by something other than EDN1. However, beyond 12 h after PGF2alpha, serum progesterone concentrations increased in EDNRA antagonist-treated animals until they were the same as saline-treated controls, whereas an EDNRB antagonist had no effect in vivo or in vitro. The EDNRA antagonist negated the antisteroidogenic actions of EDN1 but only partially abolished the actions of PGF2alpha in vitro. In contrast, the EDNRB antagonist was ineffective in abolishing antisteroidogenic actions of EDN1 and PGF2alpha. Whereas real-time RT-PCR demonstrated high expression of EDNRA and low expression of EDNRB, immunohistochemically, only EDNRA was located in small steroidogenic, endothelial, and smooth muscle cells. In summary, studies in ovine corpora lutea provided strong evidence that: 1) EDNRA, but not EDNRB, mediates antisteroidogenic actions of EDN1, 2) actions of PGF2alpha are both independent of and dependent upon mediation by EDN1, and 3) small steroidogenic cells are targets for antisteroidogenic effects of EDN1. Furthermore, the results from experiment 1 suggest that the intermediary role of EDN1 may be more important in later stages of luteal regression.     

Effects of environmental salinity on gill endothelin receptor expression in the killifish, Fundulus heteroclitus

We recently determined that rapid changes in environmental salinity alter endothelin-1 (EDN1) mRNA levels in the euryhaline killifish, Fundulus heteroclitus, so we hypothesized that EDN1 may be a local regulator of gill ion transport in teleost fishes. The purpose of the present study was to examine the effects of changes in environmental salinity on the gill endothelin receptors: EDNRA, EDNRB, and EDNRC. Using quantitative real-time PCR, we determined that after a fresh water (FW) to seawater (SW) transfer, there is a two to threefold increase in gill EDNRA and EDNRB mRNA levels. Likewise, we found a two to three fold increase in gill EDNRA and EDNRB protein concentration. In addition, killifish that have acclimated to FW for 30 days had significantly lower EDNRA mRNA and protein levels than SW killifish. ENDRA were immunolocalized to the mitochondrion-rich cells of the killifish gill, suggesting that EDN1 signaling cascades may affect MRC function. EDNRB were found throughout the gill vasculature and on lamellar pillar cells. We previously immunolocalized EDN1 to the pillar cell suggesting that EDN1 acts as an autocrine signaling molecule and potentially regulates pillar cell tone and lamellar perfusion. We conclude that EDN1 is physiologically active in the teleost gill, and regulated by environmental salinity. Future functional studies examining the physiological role of this system are necessary to completely understand EDN1 in the fish gill.     

Expression of endothelin 1 and its receptors in the hypoxic pregnant rat

Endothelin 1 (EDN1) plays a primary role in the pathophysiology of hypoxia-induced fetal growth restriction in the rat. In this study we evaluated the effects of chronic maternal hypoxia on the expression of endothelin and its receptors and on receptor binding activity in the uterus and placenta of the rat, in order to elucidate their roles in hypoxia-induced fetal growth restriction. Timed-pregnant Sprague-Dawley rats were maintained in either a normoxic or a normobaric hypoxic (12% O(2)) atmosphere from Gestational Days 18-21. Uterine and placental tissues collected on Gestational Day 21 were assayed for Edn1, Ednra, and Ednrb (endothelin receptors) mRNA expression by real-time quantitative RT-PCR, for localization of EDN1 and its receptors by immunohistochemistry, for EDNRA and EDNRB protein expression by Western blot, and for receptor binding activity by homologous competitive binding assays. EDN1 mRNA expression was significantly increased in the hypoxic placenta, but not in the uterus, compared with normoxic controls. Immunohistochemistry revealed increased EDN1 specifically in the labyrinth of the placenta. Receptor mRNA levels were not significantly affected by hypoxia, but EDNRA protein expression was significantly decreased specifically in the uterine placental beds. Receptor binding decreased significantly in response to hypoxia in all tissues investigated, compared with controls. These results suggest that chronic maternal hypoxia results in increased expression of EDN1 in the placenta but not in the uterus, and that reduced binding activity, rather than regulation of receptor expression, is a mechanism by which these tissues regulate the local hemodynamic response to increased endogenous placental EDN1 in the setting of hypoxia.     

Mutational analysis of the complex of human RNase inhibitor and human eosinophil-derived neurotoxin (RNase 2)

RNase inhibitor (RI) binds diverse proteins in the pancreatic RNase superfamily with extremely high avidity. Previous studies showed that tight binding of RNase A and angiogenin (Ang) is achieved primarily through interactions of hot spot residues in the 434-460 C-terminal segment of RI with the enzymatic active site; Asp435 of RI forms key hydrogen bonds with the catalytic lysine in both complexes, whereas the other contacts are largely distinctive. Here we have investigated the structural basis for recognition of a third ligand, eosinophil-derived neurotoxin (EDN), by single-site and multisite mutagenesis. Surprisingly, Ala replacement of Asp435 decreases affinity for EDN only by 14-fold, as compared to the several hundred-fold decreases with RNase A and Ang, and individual mutations of three other hot spot residues-Tyr434, Tyr437, and Ser460-have essentially no effect. Ala substitutions of nine additional residues, selected by examining a computational model of the RI.EDN complex, also have no marked impact. Overall, the losses in affinity for the single-residue variants examined account for only approximately 25% of the free energy of binding for the complex. However, multisite mutagenesis of RI reveals strong superadditivity of mutational effects, indicating that part of this shortfall reflects negative cooperativity. Replacement of Tyr434 together with Asp435 or Tyr437 increases K(i) by 540- and 290-fold, respectively. Thus, the C-terminal region of RI again plays an important role in ligand recognition, although probably smaller than for binding RNase A and Ang. Simultaneous substitutions of three neighboring tryptophans (261, 263, and 318) on RI attenuate affinity even more dramatically (by 4900-fold), indicating that the interactions of this RI region also contribute a considerable amount of the binding energy for the EDN complex. These findings highlight the potential importance of cooperativity in protein-protein interactions and the consequent limitations of single-site mutagenesis for assessing interface energetics.     

Effect of SNP Polymorphisms of EDN1, EDNRA,and EDNRB Gene on Ischemic Stroke

The objective of this study is to investigate the association between SNP polymorphisms of endothelin-1 (EDN1) and endothelin receptor (EDNRA and EDNRB) gene and ischemic stroke (IS) in the Chinese Han population in northern. A case–control study was introduced. We genotyped eight SNPs (rs1800541, rs2070699, and rs5370 in EDN1 gene; rs1801708, rs5333, and rs5335 in EDNRA gene; and rs3818416 and rs5351 in EDNRB gene) and calculated their polymorphic distribution in control group, IS group, and the IS subgroups. In male population, EDN1 gene rs2070699 G allele increased the incidence risk to 1.78 times (P = 0.009; OR 1.78; 95 % CI 1.15–2.75) and the risk of morbidity of rs5370 T allele carrying increased to 1.49 times (P = 0.048; OR 1.49; 95 % CI 1.00–2.21). EDNRA gene mutation rs5335 homozygous CC morbidity risk was significantly lower (P = 0.016; OR 0.52; 95 % CI 0.31–0.88). In the female population, the mutant homozygous AA cancer risk was significantly higher than G allele carriers (P = 0.019; OR 2.65; 95 % CI 1.18–6.00) on EDNRA gene rs1801708. In EDN1 gene, T allele of rs5370 and G allele of rs2070699 may be IS incidence risk factors in Northern Han male population. A allele of rs1801708 in EDNRA gene can increase the risk of IS in Northern Han women population.     

Rational engineering of a fluorescein-binding anticalin for improved ligand affinity

The anticalin FluA is an artificial lipocalin with novelspecificity for the fluorescein group, which was engineered from an insect bilin-binding protein by targeted random mutagenesis and selection. Based on the crystal structure of FluA, an attempt was made to improve the complementarity of its ligand pocket to fluorescein by rational protein design. Several side chains participating in sub-optimal interactions with the ligand were identified and replaced by residues that promised a better steric fit. As a result, the substitution of Ala45 by Ile and of Ser114 by Thr or Arg led to a tight affinity of ca. 1 nM, which is approximately 30-fold better than that of the parental anticalin. Similar to the original FluA, the improved version shows almost complete quenching of the bound ligand fluorescence. Interestingly, the quenching effect was significantly reduced when Trp129 was replaced by Tyr, thus supporting the previously postulated role of this residue, which closely packs against the bound ligand, for efficient electron transfer to the excited fluorescein. Circular dichroism spectra revealed that all variants investigated had retained the lipocalin fold. Corresponding thermal unfolding experiments confirmed similar folding stabilities, with melting temperatures ranging from 52.9 to 60.5 degrees C (i.e., for the high-affinity variant).     

Structure/function of the human glucocorticoid receptor: tyrosine 735 is important for transactivation.

Ligand-induced activation of the glucocorticoid receptor (GR) is not well understood. The GR ligand-binding domain was modeled, based on homology with the progesterone receptor. Tyrosine 735 interacts with the D ring of dexamethasone, and substitution of D ring functional groups results in partial agonist steroids with reduced ability to direct transactivation. Loss of the Tyr735 hydroxyl group by substitution to phenylalanine (Tyr735Phe) did not reduce ligand binding affinity [dissociation constant (Kd) 4.3 nM compared with Kd 4.6 nM for wild-type] and did not alter transrepression of an nuclear factor-kappaB (NF-kappaB reporter. But, there was a significant 30% reduction in maximal transactivation of a mouse mammary tumor virus (MMTV) reporter, although with an unchanged EC50 (8.6 nM compared with 6 nM). Substitution to a nonaromatic hydrophobic amino acid, valine (Tyr735Val), retained high-affinity ligand binding for dexamethasone (Kd 6 nM compared with 4.6 nM) and did not alter transrepression of NF-kappaB. However, there was a 36% reduction in MMTV activity with a right shift in EC50 (14.8 nM). The change to serine, a small polar amino acid (Tyr735Ser), caused significantly lower affinity for dexamethasone (10.4 nM). Maximal transrepression of NF-kappaB was unaltered, but the IC50 for this effect was increased. Tyr735Ser had a major shift in EC50 (118 nM) for transactivation of an MMTV reporter. Maximal transactivation of MMTV induced by the natural ligand cortisol was reduced to 60% by Tyr735Phe and Tyr735Val and was completely absent by Tyr735Ser. These data suggest that tyrosine 735 is important for ligand interpretation and transactivation.     

The structure of a doripenem‐bound OXA‐51 class D β‐lactamase variant with enhanced carbapenemase activity

OXA‐51 is a class D β‐lactamase that is thought to be the native carbapenemase of Acinetobacter baumannii. Many variants of OXA‐51 containing active site substitutions have been identified from A. baumannii isolates, and some of these substitutions increase hydrolytic activity toward carbapenem antibiotics. We have determined the high‐resolution structures of apo OXA‐51 and OXA‐51 with one such substitution (I129L) with the carbapenem doripenem trapped in the active site as an acyl‐intermediate. The structure shows that acyl‐doripenem adopts an orientation very similar to carbapenem ligands observed in the active site of OXA‐24/40 (doripenem) and OXA‐23 (meropenem). In the OXA‐51 variant/doripenem complex, the indole ring of W222 is oriented away from the doripenem binding site, thereby eliminating a clash that is predicted to occur in wildtype OXA‐51. Similarly, in the OXA‐51 variant complex, L129 adopts a different rotamer compared to I129 in wildtype OXA‐51. This alternative position moves its side chain away from the hydroxyethyl moiety of doripenem and relieves another potential clash between the enzyme and carbapenem substrates. Molecular dynamics simulations of OXA‐51 and OXA‐51 I129L demonstrate that compared to isoleucine, a leucine at this position greatly favors a rotamer that accommodates the ligand. These results provide a molecular justification for how this substitution generates enhanced binding affinity for carbapenems, and therefore helps explain the prevalence of this substitution in clinical OXA‐51 variants.     

Imputation of Exome Sequence Variants into Population- Based Samples and Blood-Cell-Trait-Associated Loci in African Americans: NHLBI GO Exome Sequencing Project

Researchers have successfully applied exome sequencing to discover causal variants in selected individuals with familial, highly penetrant disorders. We demonstrate the utility of exome sequencing followed by imputation for discovering low-frequency variants associated with complex quantitative traits. We performed exome sequencing in a reference panel of 761 African Americans and then imputed newly discovered variants into a larger sample of more than 13,000 African Americans for association testing with the blood cell traits hemoglobin, hematocrit, white blood count, and platelet count. First, we illustrate the feasibility of our approach by demonstrating genome-wide-significant associations for variants that are not covered by conventional genotyping arrays; for example, one such association is that between higher platelet count and an MPL c.117G>T (p.Lys39Asn) variant encoding a p.Lys39Asn amino acid substitution of the thrombpoietin receptor gene (p = 1.5 × 10⁻¹¹). Second, we identified an association between missense variants of LCT and higher white blood count (p = 4 × 10⁻¹³). Third, we identified low-frequency coding variants that might account for allelic heterogeneity at several known blood cell-associated loci: MPL c.754T>C (p.Tyr252His) was associated with higher platelet count; CD36 c.975T>G (p.Tyr325^∗) was associated with lower platelet count; and several missense variants at the α-globin gene locus were associated with lower hemoglobin. By identifying low-frequency missense variants associated with blood cell traits not previously reported by genome-wide association studies, we establish that exome sequencing followed by imputation is a powerful approach to dissecting complex, genetically heterogeneous traits in large population-based studies.     

Mutations remote from the human gonadotropin-releasing hormone (GnRH) receptor-binding sites specifically increase binding affinity for GnRH II but not GnRH I: evidence for ligand-selective, receptor-active conformations

The human gonadotropin-releasing hormone (GnRH) receptor is evolutionarily configured for high affinity binding of GnRH I ([Tyr(5),Leu(7),Arg(8)]GnRH) but at lower affinity for GnRH II ([His(5),Trp(7),Tyr(8)]GnRH). GnRH I is more potent in the activation of the G(q/11) protein in the gonadotrope; however, GnRH II is more potent in the stimulation of apoptosis and antiproliferative effects through activating G(i) protein-mediated signaling, implying that GnRH I and II selectively stabilize different receptor-active conformations that preferentially couple to different signaling pathways. Receptor activation involves ligand induction or conformational selection, but the molecular basis of the communication between ligand-binding sites and receptor allosteric sites remains unclear. We have sought conformational coupling between receptor-ligand intermolecular interactions and intramolecular interaction networks in the human GnRH receptor by mutating remote residues that induce differential ligand binding affinity shifts for GnRH I and II. We have demonstrated that certain Ala mutations in the intracellular segments of transmembrane domains 3 (Met(132)), 5 (Met(227)), 6 (Phe(272) and Phe(276)), and 7 (Ile(322) and Tyr(323)) of the human GnRH receptor allosterically increased ligand binding affinity for GnRH II but had little effect on GnRH I binding affinity. We examined the role of the three amino acids that differ in these two ligands, and we found that Tyr(8) in GnRH II plays a dominant role for the increased affinity of the receptor mutants for GnRH II. We propose that creation of a high affinity binding site for GnRH II accompanies receptor conformational changes, i.e."induced fit" or "conformational selection," mainly determined by the intermolecular interactions between Tyr(8) and the receptor contact residues, which can be facilitated by disruption of particular sets of receptor-stabilizing intramolecular interactions. The findings suggest that GnRH I and II binding may selectively stabilize different receptor-active conformations and therefore different ligand-induced selective signaling described previously for these ligands.     

Two closely spaced missense COL3A1 variants in cis cause vascular Ehlers‐Danlos syndrome in one large Chinese family

Vascular Ehlers‐Danlos syndrome (vEDS) is a rare and severe hereditary connective tissue disease arising from a mutation in the type III collagen alpha I chain (COL3A1) gene, with a poor prognosis due to exceptional vascular ruptures and premature death. Herein, starting from a 36‐year‐old Chinese male patient with a complaint of upper abdominal pain, we collected clinical data of and performed a genetic analysis of a total of 20 family members. We identified two closely spaced COL3A1 missense variants in cis, p.Leu734Phe (c.2199_2200TC>AT) and p.Gly741Ser (c.2221G>A), as the cause of vEDS in this family. p.Gly741Ser, a glycine substitution mutation, has been previously reported, whereas p.Leu734Phe, a non‐glycine substitution mutation, is novel. We analysed their independent and combined effects on the COL3A1 level in transfected skin fibroblast cells by means of Western blotting. We found that both variants independently led to a reduced COL3A1 level and, when combined, led to an even more reduced COL3A1 level compared to the wild type. Thus, each missense variant can be independently classified as a pathogenic variant, albeit with a synergetic effect when occurring together. Moreover, our genetic findings provide an explanation for four previous sudden deaths and identified two high‐risk carriers in the family.     

Tyrosine 220 in the 5th transmembrane domain of the neuromedin B receptor is critical for the high selectivity of the peptoid antagonist PD168368

Peptoid antagonists are increasingly being described for G protein-coupled receptors; however, little is known about the molecular basis of their binding. Recently, the peptoid PD168368 was found to be a potent selective neuromedin B receptor (NMBR) antagonist. To investigate the molecular basis for its selectivity for the NMBR over the closely related receptor for gastrin-releasing peptide (GRPR), we used a chimeric receptor approach and a site-directed mutagenesis approach. Mutated receptors were transiently expressed in Balb 3T3. The extracellular domains of the NMBR were not important for the selectivity of PD168368. However, substitution of the 5th upper transmembrane domain (uTM5) of the NMBR by the comparable GRPR domains decreased the affinity 16-fold. When the reverse study was performed by substituting the uTM5 of NMBR into the GRPR, a 9-fold increase in affinity occurred. Each of the 4 amino acids that differed between NMBR and GRPR in the uTM5 region were exchanged, but only the substitution of Phe(220) for Tyr in the NMBR caused a decrease in affinity. When the reverse study was performed to attempt to demonstrate a gain of affinity in the GRPR, the substitution of Tyr(219) for Phe caused an increase in affinity. These results suggest that the hydroxyl group of Tyr(220) in uTM5 of NMBR plays a critical role for high selectivity of PD168368 for NMBR over GRPR. Receptor and ligand modeling suggests that the hydroxyl of the Tyr(220) interacts with nitrophenyl group of PD168368 likely primarily by hydrogen bonding. This result shows the selectivity of the peptoid PD168368, similar to that reported for numerous non-peptide analogues with other G protein-coupled receptors, is primarily dependent on interaction with transmembrane amino acids.     

Biosynthesis of bacterial glycogen. Use of site-directed mutagenesis to probe the role of tyrosine 114 in the catalytic mechanism of ADP-glucose synthetase from Escherichia coli

Previous covalent modification studies showed that tyrosine 114 of Escherichia coli ADP-glucose synthetase is involved in substrate binding (Lee, Y. M., and Preiss, J. (1986) J. Biol. Chem. 261, 1058-1064). We have prepared, via site-directed mutagenesis, an E. coli ADP-glucose synthetase variant (Phe114) containing a Tyr114 to Phe substitution in order to test whether the phenolic hydroxyl group plays a critical role in catalysis. Kinetic characterization of Phe114 ADP-glucose synthetase indicates that the Tyr114 hydroxyl is not obligatory for the enzyme catalysis. However, the variant enzyme showed altered properties. It showed a decreased apparent affinity for the substrates. The variant enzyme showed less than 2-fold activation by 5 mM fructose 1,6-bisphosphate in the ADP-glucose synthesis direction. In contrast, in the pyrophosphorolysis direction, the mutant enzyme showed about a 30-fold activation by 5 mM fructose 1,6-bisphosphate. The variant enzyme is heat-labile compared to wild type enzyme. It lost about 60% enzyme activity on incubation at 65 degrees C for 5 min in the presence of 30 mM Pi. The wild type enzyme is stable under these conditions. The results indicate that tyrosine 114 is involved directly or indirectly in enzyme catalysis, but is not obligatory for the enzyme catalysis. Conversion of Tyr114 to Phe also alters the regulatory properties of the enzyme with respect to activation by fructose-1,6-P2 and inhibition by AMP.     

Penetrance of Hemochromatosis in HFE Genotypes Resulting in p.Cys282Tyr and p.[Cys282Tyr];[His63Asp] in the eMERGE Network

Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder associated with pathogenic HFE variants, most commonly those resulting in p.Cys282Tyr and p.His63Asp. Recommendations on returning incidental findings of HFE variants in individuals undergoing genome-scale sequencing should be informed by penetrance estimates of HH in unselected samples. We used the eMERGE Network, a multicenter cohort with genotype data linked to electronic medical records, to estimate the diagnostic rate and clinical penetrance of HH in 98 individuals homozygous for the variant coding for HFE p.Cys282Tyr and 397 compound heterozygotes with variants resulting in p.[His63Asp];[Cys282Tyr]. The diagnostic rate of HH in males was 24.4% for p.Cys282Tyr homozygotes and 3.5% for compound heterozygotes (p < 0.001); in females, it was 14.0% for p.Cys282Tyr homozygotes and 2.3% for compound heterozygotes (p < 0.001). Only males showed differences across genotypes in transferrin saturation levels (100% of homozygotes versus 37.5% of compound heterozygotes with transferrin saturation > 50%; p = 0.003), serum ferritin levels (77.8% versus 33.3% with serum ferritin > 300 ng/ml; p = 0.006), and diabetes (44.7% versus 28.0%; p = 0.03). No differences were found in the prevalence of heart disease, arthritis, or liver disease, except for the rate of liver biopsy (10.9% versus 1.8% [p = 0.013] in males; 9.1% versus 2% [p = 0.035] in females). Given the higher rate of HH diagnosis than in prior studies, the high penetrance of iron overload, and the frequency of at-risk genotypes, in addition to other suggested actionable adult-onset genetic conditions, opportunistic screening should be considered for p.[Cys282Tyr];[Cys282Tyr] individuals with existing genomic data.     

Variants of endothelin-1 and its receptors in atopic asthma.

Endothelin-1 (ET-1) is a 21 amino acid peptide released from several types of bronchial cells. It operates through two types of receptors, type A(ET-RA) and type B(ET-RB) and has various activities in the pathophysiology of atopic asthma. These genes are localised on different chromosomes where genome-wide searches have identified linkage for atopic asthma, thus supporting the candidacy of ET-1 and its receptors for atopic asthma. A genetic association study was performed with variants of these three genes in both British (n = 300) and Japanese populations (n = 200). No significant association was found between variants of EDN1 and EDNRB genes, and atopic asthma in either population. However, variants of EDNRA gene showed a marginal association with atopy [odds = 0.39(95% CI: 0.17-0.89), p = 0.022, Pc = 0.066], especially with antigen specific IgE levels [odds = 0.31 (95% CI: 0.20-0.77), p = 0.006, Pc = 0.018] in the British population. These findings suggest that EDNRA is a major candidate locus for atopy on chromosome 4.