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Umami is one of the 5 basic taste qualities. The umami taste of L-glutamate can be drastically enhanced by 5' ribonucleotides and the synergy is a hallmark of this taste quality. The umami taste receptor is a heteromeric complex of 2 class C G-protein-coupled receptors, T1R1 and T1R3. Here we elucidate the molecular mechanism of the synergy using chimeric T1R receptors, site-directed mutagenesis, and molecular modeling. We propose a cooperative ligand-binding model involving the Venus flytrap domain of T1R1, where L-glutamate binds close to the hinge region, and 5' ribonucleotides bind to an adjacent site close to the opening of the flytrap to further stabilize the closed conformation. This unique mechanism may apply to other class C G-protein-coupled receptors.

Positive allosteric modulators of the human sweet taste receptor have been developed as a new way of reducing dietary sugar intake. Besides their potential health benefit, the sweet taste enhancers are also valuable tool molecules to study the general mechanism of positive allosteric modulations of T1R taste receptors. Using chimeric receptors, mutagenesis, and molecular modeling, we reveal how these sweet enhancers work at the molecular level. Our data argue that the sweet enhancers follow a similar mechanism as the natural umami taste enhancer molecules. Whereas the sweeteners bind to the hinge region and induce the closure of the Venus flytrap domain of T1R2, the enhancers bind close to the opening and further stabilize the closed and active conformation of the receptor.

To identify molecules that could enhance sweetness perception, we undertook the screening of a compound library using a cell-based assay for the human sweet taste receptor and a panel of selected sweeteners. In one of these screens we found a hit, SE-1, which significantly enhanced the activity of sucralose in the assay. At 50 μM, SE-1 increased the sucralose potency by >20-fold. On the other hand, SE-1 exhibited little or no agonist activity on its own. SE-1 effects were strikingly selective for sucralose. Other popular sweeteners such as aspartame, cyclamate, and saccharin were not enhanced by SE-1 whereas sucrose and neotame potency were increased only by 1.3- to 2.5-fold at 50 μM. Further assay-guided chemical optimization of the initial hit SE-1 led to the discovery of SE-2 and SE-3, selective enhancers of sucralose and sucrose, respectively. SE-2 (50 μM) and SE-3 (200 μM) increased sucralose and sucrose potencies in the assay by 24- and 4.7-fold, respectively. In human taste tests, 100 μM of SE-1 and SE-2 allowed for a reduction of 50% to >80% in the concentration of sucralose, respectively, while maintaining the sweetness intensity, and 100 μM SE-3 allowed for a reduction of 33% in the concentration of sucrose while maintaining the sweetness intensity. These enhancers did not exhibit any sweetness when tasted on their own. Positive allosteric modulators of the human sweet taste receptor could help reduce the caloric content in food and beverages while maintaining the desired taste.

The sweet taste receptor is rather unique, recognizing a diverse repertoire of natural or synthetic ligands, with a surprisingly large structural diversity, and with potencies stretching over more than six orders of magnitude. Yet, it is not clear if different cell-based assays can faithfully report the relative potencies and efficacies of these molecules. Indeed, up to now, sweet taste receptor agonists have been almost exclusively characterized using cell-based assays developed with overexpressed and promiscuous G proteins. This non-physiological coupling has allowed the quantification of receptor activity via phospholipase C activation and calcium mobilization measurements in heterologous cells on a FLIPR system, for example. Here, we developed a novel assay for the human sweet taste receptor where endogenous G proteins and signaling pathways are recruited by the activated receptor. The effects of several sweet taste receptor agonists and other types of modulators were recorded by measuring changes in dynamic mass redistribution (DMR) using an Epic ® reader. Potency and efficacy values obtained in the DMR assay were compared to those results obtained with the classical FLIPR assay. Results demonstrate that for some ligands, the two assay systems provide similar information. However, a clear bias for the FLIPR assay was observed for one third of the agonists evaluated, suggesting that the use of non-physiological coupling may influence the potency and efficacy of sweet taste receptor ligands. Replacing the promiscuous G protein with a chimeric G protein containing the C-terminal tail 25 residues of the physiologically relevant G protein subunit Gα gustducin reduced or abrogated bias.

Globozoospermia is a rare phenotype of primary male infertility inducing the production of round-headed spermatozoa without acrosome. Anomalies of DPY19L2 account for 50–70% of all cases and the entire deletion of the gene is by far the most frequent defect identified. Here, we present a large cohort of 69 patients with 20–100% of globozoospermia. Genetic analyses including multiplex ligation-dependent probe amplification, Sanger sequencing and whole-exome sequencing identified 25 subjects with a homozygous DPY19L2 deletion (36%) and 14 carrying other DPY19L2 defects (20%). Overall, 11 deleterious single-nucleotide variants were identified including eight novel and three already published mutations. Patients with a higher rate of round-headed spermatozoa were more often diagnosed and had a higher proportion of loss of function anomalies, highlighting a good genotype phenotype correlation. No gene defects were identified in patients carrying < 50% of globozoospermia while diagnosis efficiency rose to 77% for patients with > 50% of globozoospermia. In addition, results from whole-exome sequencing were scrutinized for 23 patients with a DPY19L2 negative diagnosis, searching for deleterious variants in the nine other genes described to be associated with globozoospermia in human (C2CD6, C7orf61, CCDC62, CCIN, DNAH17, GGN, PICK1, SPATA16, and ZPBP1). Only one homozygous novel truncating variant was identified in the GGN gene in one patient, confirming the association of GGN with globozoospermia. In view of these results, we propose a novel diagnostic strategy focusing on patients with at least 50% of globozoospermia and based on a classical qualitative PCR to detect DPY19L2 homozygous deletions. In the absence of the latter, we recommend to perform whole-exome sequencing to search for defects in DPY19L2 as well as in the other previously described candidate genes.

Background Germline mosaicism is considered to be a rare event. However, its occurrence is underestimated due to the limited availability of germ cells. The genomic variations that underlie this phenomenon comprise single nucleotide polymorphism (SNPs), copy number variations (CNVs) and aneuploidies. In the case of CNVs, deletions are more frequent in the paternal germline while duplications are more commonly maternal in origin. Germline mosaicism increases with paternal age as the risk of SNPs increase with the number of germ cell divisions. We here report a case of germline mosaicism in the spermatozoa of a donor that resulted in one pathological pregnancy. Results Straws from the same sperm donor were provided to seven recipient couples, resulting in four pregnancies. Second trimester ultrasound analysis revealed bilateral talipes equinovarus associated with growth retardation in one of these pregnancies. Array-comparative genomic hybridization (CGH) carried out after amniocentesis revealed a 4 Mb deletion in the 7q32.1q33 region. The blood karyotypes and array-CGHs were normal in the mother, as well as in the donor. However, the microsatellite profile indicated a paternal origin. Fluorescent in situ hybridization (FISH) analysis of the donor’s spermatozoa revealed the same chromosomal rearrangements in 12% of the spermatozoa population. Due to the documented risk of mental retardation associated with genomic rearrangements in the same region, the couple decided to terminate the pregnancy. Amniocentesis was performed in the other couples, which yielded normal FISH analysis results. Conclusions Several cases of germline mosaicism have been reported to date, but their frequency is probably underestimated. Moreover, it is important to note that germline mosaicism cannot be ruled out by conventional cytogenetic screening of blood cells. This case highlights the need for close follow-up of every pregnancy obtained through gamete donation, given that the occurrence of germline mosaicism may have major consequences when multiple pregnancies are obtained concomitantly.

Résumé Contexte La mise en évidence d’une mosaïque germinale est. un événement rare mais probablement sous-estimé du fait de l’accès limité aux cellules germinales. Les variations génomiques caractéristiques de ce phénomène peuvent être des single nucleotide polymorphismes (SNPs), des copy number variations (CNVs) ou des aneuploïdies. Dans le cas des CNVs, les délétions sont plus fréquentes dans la lignée germinale paternelle tandis que les duplications sont plus fréquemment d’origine maternelle. Le risque de mosaïcisme germinal augmente avec l’âge paternel de part une augmentation du risque de SNPs associée à la division constante des cellules germinales pendant toute la vie d’un homme. Nous rapportons ici un cas de mosaïque germinale chez un donneur de spermatozoïdes ayant entraîné la survenue d’une grossesse pathologique. Résultats Les paillettes d’un même donneur de spermatozoïdes ont été attribuées à sept couples receveurs permettant l’obtention de quatre grossesses évolutives. Pour l’une d’entre elle, l’échographie du deuxième trimestre a permis d’identifier chez le fœtus des pieds bots associés à un retard de croissance intra utérin. L’analyse par hybridation génomique comparative (CGH)-array après amniocentèse a révélé une délétion de 4 MB dans la région 7q32.1q33. Les caryotypes sanguins et les analyses par CGH-array étaient normaux chez la mère et le donneur. Cependant les profils de microsatellites ont montré une origine paternelle du chromosome délété. Une analyse par fluorescent in situ hybridization (FISH) des spermatozoïdes du donneur a révélé la présence de la même délétion dans 12% des spermatozoïdes étudiés. Etant donné le risque de retard mental associé à des remaniements chromosomiques dans cette même région, le couple a préféré interrompre la grossesse. Une amniocentèse a été réalisée pour les autres grossesse en cours et n’a retrouvé aucune anomalie. Conclusions Plusieurs cas de mosaïques germinales ont été rapportés mais leur fréquence réelle reste probablement sous-estimée. En effet, un mosaïcisme germinal ne peut être détecté par les techniques de cytogénétique conventionnelle sur sang. Ce cas illustre la nécessité d’un suivi en temps réel des grossesses obtenues par don de spermatozoïdes étant donné que la survenue d’une grossesse pathologique peut avoir un retentissement sur les autres grossesses issues du même donneur.

To assess the frequency of certain etiologies in fetal echogenic bowel and the related pregnancy outcome. A retrospective study including 215 cases. Ultrasound findings included bowel abnormality, malformations, growth retardation and Doppler abnormalities. Amniocentesis was performed in 196 cases to screen for abnormalities in karyotype, cystic fibrosis gene and infection. The color of amniotic fluid and a history of first-trimester bleeding were also noted. Outcome according to different etiologies was reported. 112 cases (57%) had a known etiology, which included chromosomal abnormality (7%), infection (4%), cystic fibrosis (1.5%), bowel abnormality (3%), bleeding or stained amniotic fluid (11%), Doppler abnormality (14%), malformation (16%) and miscellaneous (0.5%). Pregnancy was terminated in 39 cases and fetal demise complicated one third of the cases (13) of severe growth retardation. Fetal echogenic bowel should be investigated and a careful follow-up is necessary if there is associated Doppler perturbation or growth retardation.

Objective: To assess the frequency of certain etiologies in fetal echogenic bowel and the related pregnancy outcome. Study Design: A retrospective study including 215 cases. Ultrasound findings included bowel abnormality, malformations, growth retardation and Doppler abnormalities. Amniocentesis was performed in 196 cases to screen for abnormalities in karyotype, cystic fibrosis gene and infection. The color of amniotic fluid and a history of first-trimester bleeding were also noted. Outcome according to different etiologies was reported. Results: 112 cases (57%) had a known etiology, which included chromosomal abnormality (7%), infection (4%), cystic fibrosis (1.5%), bowel abnormality (3%), bleeding or stained amniotic fluid (11%), Doppler abnormality (14%), malformation (16%) and miscellaneous (0.5%). Pregnancy was terminated in 39 cases and fetal demise complicated one third of the cases (13) of severe growth retardation. Conclusion: Fetal echogenic bowel should be investigated and a careful follow-up is necessary if there is associated Doppler perturbation or growth retardation.