Midgut neuroendocrine tumor patients have a depleted gut microbiome with a discriminative signature.

RATIONALE: When compared to other types of cancer, the prevalence of midgut neuroendocrine tumors (NET) has disproportionally increased over the past decades. To date, there has been very little progress in discovering (epi)genetic drivers and treatment options for these tumors. Recent microbiome research has revealed that enteroendocrine cells communicate with the intestinal microbiome and has provided novel treatment targets for various other cancer types. Hence, our aim was to analyze the role of the gut microbiome in midgut NET patients. METHODS: Fecal samples, prospectively collected from patients and control subjects, were analyzed with next generation 16S sequencing. Patients with neuroendocrine carcinomas and recent antibiotics use were excluded. Relevant variables were extracted from questionnaires and electronic health records. Microbial composition was compared between patients and controls as well as between groups within the patient cohort. RESULTS: 87 midgut NET patients and 95 controls were included. Midgut NET patients had a less rich and diverse gut microbiome than controls (p < 0.001). Moreover, we identified 31 differentially abundant species and a gut microbial signature consisting of 17 species that was predictive of midgut NET presence with an area under the receiver operating characteristic curve of 0.863. Gut microbial composition was not directly associated with the presence of the carcinoid syndrome, tumor grade or multifocality. Nonetheless, we did observe a potential link between microbial diversity and the presence of carcinoid syndrome symptoms within the subset of patients with elevated 5-hydroxyindolacetic acid levels. CONCLUSION: Midgut NET patients have an altered gut microbiome which suggests a role in NET development and could provide novel targets for microbiome-based diagnostics and therapeutics.

Prevalence of and risk factors for extended-spectrum beta-lactamase genes carriership in a population-based cohort of middle-aged and elderly.

INTRODUCTION: Increasing resistance to beta-lactam antibiotics is an alarming development worldwide. Fecal carriership of TEM, SHV, CTX-M and CMY was studied in a community-dwelling population of middle-aged and elderly individuals. PATIENTS AND METHODS: Feces was obtained from individuals of the Rotterdam Study. Carriership of the TEM, SHV, CTX-M and CMY genes was determined using real-time polymerase chain reaction (qPCR). Possible associations were investigated between carriership of these genes and several risk factors, such as the use of antimicrobial drugs, diabetes mellitus, protein pump inhibitor (PPI) use, travelling, the composition of the gut microbiota, and intake of certain foods. RESULTS: The most prevalent gene was TEM (53.0%), followed by SHV (18.4%), CTX-M (5.4%) and CMY (3.6%). Use of penicillins with extended spectrum was associated with TEM carriership, whereas use of macrolides and lincosamides was associated with TEM and SHV carriership. Interestingly, use of PPIs was associated with a higher prevalence of carriership of TEM, SHV and CMY (TEM: odds ratio [OR] 1.34; 95% confidence interval [CI] 1.05-1.77; SHV: OR 2.17; 95%CI 1.55-2.87; CMY: OR 2.26; 95%CI 1.23-4.11). Furthermore, associations were found between the richness and composition of the gut microbiota and TEM and SHV carriership. CONCLUSIONS: The prevalence of carriership of TEM was substantial, but the prevalence of carriership of the extended-spectrum ß-lactamase gene, CTX-M and the AmpC ß-lactamase gene, CMY was relatively low in this community-dwelling, population-based cohort. The composition of the microbiota might play a role in the retention of resistance genes, but future studies are necessary to further elucidate this relationship.

Long-term effects of antimicrobial drugs on the composition of the human gut microbiota.

INTRODUCTION: Antimicrobial drugs are known to have effects on the human gut microbiota. We studied the long-term temporal relationship between several antimicrobial drug groups and the composition of the human gut microbiota determined in feces samples. METHODS: Feces samples were obtained from a community-dwelling cohort of middle-aged and elderly individuals (Rotterdam Study). Bacterial DNA was isolated and sequenced using V3/V4 16 S ribosomal RNA sequencing (Illumina MiSeq). The time between the last prescription of several antimicrobial drug groups and the day of sampling was categorized into 0-12, 12-24, 24-48 and >48 months. The effects of the antimicrobial drug groups on the Shannon alpha-diversity (diversity), the Bray-Curtis beta-diversity (community structure), the Firmicutes/Bacteroidetes (F/B) ratio and individual genera were determined. RESULTS: We studied the gut microbiota of 1413 individuals (57.5% female, median age 62.6 years). The alpha-diversity was significantly lower up to 4 years after prescriptions of macrolides and lincosamides. It was also lower in the first year after the use of beta-lactams. The community structure (beta-diversity) of the microbiota was significantly different up to 4 years for macrolides and lincosamides, the first year for beta-lactams and at least the first year for quinolones. For the F/B ratio, drugs with a high anaerobic activity shifted the ratio toward Firmicutes in the first year whereas other antimicrobial drugs shifted the ratio toward Bacteroidetes. CONCLUSION: Use of antimicrobial drugs is associated with a shift in the composition of the gut microbiota.These effects differ in strength and duration, depending on the antimicrobial drug group used. These findings should be considered when prescribing antimicrobial drugs.

The effect of antimicrobial drug use on the composition of the genitourinary microbiota in an elderly population.

BACKGROUND: The urinary tract is inhabited by a diversity of microorganisms, known as the genitourinary microbiota. Here, we investigated the association between the use of antimicrobial drugs and the composition of the genitourinary microbiota. RESULTS: Clean-catch urinary samples were collected from 27 participants of the Rotterdam Study. Bacterial DNA was extracted and the 16S ribosomal RNA gene variable regions V3 and V4 were analyzed using Illumina sequencing. 23 of the 27 participants were included in the analysis. The population consisted of 10 men and 13 women with a mean age of 75 ± 3 years. The time between the last prescription of an antimicrobial drug and sampling was determined and categorized. The use of antimicrobial drugs prior to urine sampling was associated with statistically significant differences in the beta-diversity of the genitourinary microbiota. No association was found between antimicrobial drug use and the alpha-diversity of the genitourinary microbiota. Operational Taxonomic Units (OTUs) that were lowest in participants who used antimicrobial drug belonged to Lactobacillus and Finegoldia. In contrast, an OTU belonging to the genus Parabacteroides had higher abundances. Also, an OTU belonging to the species E.coli was higher in the participants who used antimicrobial drugs. CONCLUSION: Prior use of antimicrobial drugs is associated with a different composition of the genitourinary microbiota. Our results might indicate a persisting effect of antimicrobial drugs on the composition of the microbiota, but reverse causality cannot be ruled out. Future studies are needed to differentiate between two possibilities. Genitourinary dysbiosis could be the result of antimicrobial drug use or genitourinary dysbiosis could be a risk factor for urinary tract infections resulting in increased use of antimicrobial drugs. This may have important implications for treatment and prevention of (recurrent) UTIs.

Observational multi-centre, prospective study to characterize novel pathogen-and host-related factors in hospitalized patients with lower respiratory tract infections and/or sepsis - the "TAILORED-Treatment" study.

BACKGROUND: The emergence and spread of antibiotic resistant micro-organisms is a global concern, which is largely attributable to inaccurate prescribing of antibiotics to patients presenting with non-bacterial infections. The use of 'omics' technologies for discovery of novel infection related biomarkers combined with novel treatment algorithms offers possibilities for rapidly distinguishing between bacterial and viral infections. This distinction can be particularly important for patients suffering from lower respiratory tract infections (LRTI) and/or sepsis as they represent a significant burden to healthcare systems. Here we present the study details of the TAILORED-Treatment study, an observational, prospective, multi-centre study aiming to generate a multi-parametric model, combining host and pathogen data, for distinguishing between bacterial and viral aetiologies in children and adults with LRTI and/or sepsis. METHODS: A total number of 1200 paediatric and adult patients aged 1 month and older with LRTI and/or sepsis or a non-infectious disease are recruited from Emergency Departments and hospital wards of seven Dutch and Israeli medical centres. A panel of three experienced physicians adjudicate a reference standard diagnosis for all patients (i.e., bacterial or viral infection) using all available clinical and laboratory information, including a 28-day follow-up assessment. Nasal swabs and blood samples are collected for multi-omics investigations including host RNA and protein biomarkers, nasal microbiota profiling, host genomic profiling and bacterial proteomics. Simplified data is entered into a custom-built database in order to develop a multi-parametric model and diagnostic tools for differentiating between bacterial and viral infections. The predictions from the model will be compared with the consensus diagnosis in order to determine its accuracy. DISCUSSION: The TAILORED-Treatment study will provide new insights into the interplay between the host and micro-organisms. New host- or pathogen-related biomarkers will be used to generate a multi-parametric model for distinguishing between bacterial and viral infections. This model will be helpful to better guide antimicrobial therapy for patients with LRTI and sepsis. This study has the potential to improve patient care, reduce unnecessary antibiotic prescribing and will contribute positively to institutional, national and international healthcare economics. TRIAL REGISTRATION: NCT02025699 . Registration Date: January, 1, 2014.

A rare missense variant in RCL1 segregates with depression in extended families.

Depression is the most prevalent psychiatric disorder with a complex and elusive etiology that is moderately heritable. Identification of genes would greatly facilitate the elucidation of the biological mechanisms underlying depression, however, its complex etiology has proved to be a major bottleneck in the identification of its genetic risk factors, especially in genome-wide association-like studies. In this study, we exploit the properties of a genetic isolate and its family-based structure to explore whether relatively rare exonic variants influence the burden of depressive symptoms in families. Using a multistep approach involving linkage and haplotype analyses followed by exome sequencing in the Erasmus Rucphen Family (ERF) study, we identified a rare (minor allele frequency (MAF)=1%) missense c.1114C>T mutation (rs115482041) in the RCL1 gene segregating with depression across multiple generations. Rs115482041 showed significant association with depressive symptoms (N=2393, ßT-allele=2.33, P-value=1 × 10-4) and explained 2.9% of the estimated genetic variance of depressive symptoms (22%) in ERF. Despite being twice as rare (MAF<0.5%), c.1114C>T showed similar effect and significant association with depressive symptoms in samples from the independent population-based Rotterdam study (N=1604, ßT-allele=3.60, P-value=3 × 10-2). A comparison of RCL1 expression in human and mouse brain revealed a striking co-localization of RCL1 with the layer 1 interlaminar subclass of astrocytes found exclusively in higher-order primates. Our findings identify RCL1 as a novel candidate gene for depression and offer insights into mechanisms through which RCL1 may be relevant for depression.

Exome-sequencing in a large population-based study reveals a rare Asn396Ser variant in the LIPG gene associated with depressive symptoms.

Despite a substantial genetic component, efforts to identify common genetic variation underlying depression have largely been unsuccessful. In the current study we aimed to identify rare genetic variants that might have large effects on depression in the general population. Using high-coverage exome-sequencing, we studied the exonic variants in 1265 individuals from the Rotterdam study (RS), who were assessed for depressive symptoms. We identified a missense Asn396Ser mutation (rs77960347) in the endothelial lipase (LIPG) gene, occurring with an allele frequency of 1% in the general population, which was significantly associated with depressive symptoms (P-value=5.2 × 10-08, ß=7.2). Replication in three independent data sets (N=3612) confirmed the association of Asn396Ser (P-value=7.1 × 10-03, ß=2.55) with depressive symptoms. LIPG is predicted to have enzymatic function in steroid biosynthesis, cholesterol biosynthesis and thyroid hormone metabolic processes. The Asn396Ser variant is predicted to have a damaging effect on the function of LIPG. Within the discovery population, carriers also showed an increased burden of white matter lesions (P-value=3.3 × 10-02) and a higher risk of Alzheimer's disease (odds ratio=2.01; P-value=2.8 × 10-02) compared with the non-carriers. Together, these findings implicate the Asn396Ser variant of LIPG in the pathogenesis of depressive symptoms in the general population.

A transductionally retargeted adenoviral vector for virotherapy of Her2/neu-expressing prostate cancer.

The efficacy of adenovirus (Ad)-based gene therapy of solid tumors, such as prostate cancer, is limited. One of the many problems is that the virus infects many different cell types in the body, resulting in high toxicity, whereas the target cancer cells are often less prone to wild-type Ad infection. Our aim was to develop genetically de- and retargeted Ad vectors to reduce off-target effects and increase target infection for prostate cancer. We have previously reported an Ad5 vector specific for the cancer-associated receptor Her2/neu, created by inserting Her2/neu-reactive Affibody(®) molecules (ZH) into the HI loop of a coxsackievirus and adenovirus receptor binding-ablated fiber (Ad[ZH/1]). In addition to virus retargeting to Her2/neu, this virus was further modified from wild-type Ad by changing the RGD motif in the penton base to EGD and by substitution of the KKTK motif in the third shaft repeat to RKSK, resulting in the vector Ad[ZH/3]. The ZH-containing vectors could be produced to high titers and were specific for their target, resulting in efficient infection and killing of Her2/neu-positive androgen-dependent PC346C prostate cancer cells in vitro. Here we show that the oncolytic Ad[ZH/3] vector significantly prolonged survival time and reduced serum prostate-specific antigen levels in an orthotopic prostate tumor model in nude mice to the same extent as wild-type Ad5. Our results show that Her2/neu targeting using Ad-based vectors for prostate cancer is feasible and may serve as a basis for the development of gene therapy of human prostate cancer as well as other Her2/neu-expressing cancers.

Disconnecting the yin and yang relation of epidermal growth factor receptor (EGFR)-mediated delivery: a fully synthetic, EGFR-targeted gene transfer system avoiding receptor activation.

The epidermal growth factor receptor (EGFR) is upregulated within a high percentage of solid tumors and hence is an attractive target for tumor-targeted therapies including gene therapy. The natural EGFR ligand epidermal growth factor (EGF) has been used for this purpose, despite the risk of mitogenic effects due to EGFR activation. We have developed a fully synthetic, EGFR-targeted gene delivery system based on PEGylated linear polyethylenimine (LPEI), allowing evaluation of different EGFR-binding peptides in terms of transfection efficiency and EGFR activation. Peptide sequences directly derived from the human EGF molecule enhanced transfection efficiency with concomitant EGFR activation. Only the EGFR-binding peptide GE11, which has been identified by phage display technique, showed specific enhancement of transfection on EGFR-overexpressing tumor cells including glioblastoma and hepatoma, but without EGFR activation. EGFR targeting led to high levels of cell association of fluorescently labeled polyplexes after only 30 min of incubation. EGF pretreatment of cells induced enhanced cellular internalization of all polyplex types tested, pointing at generally enhanced macropinocytosis. EGF polyplexes diminished cell surface expression of EGFR for up to 4 hr, whereas GE11 polyplexes did not. In a clinically relevant orthotopic prostate cancer model, intratumorally injected GE11 polyplexes were superior in inducing transgene expression when compared with untargeted polyplexes.

Virus specific immune responses after human neoadjuvant adenovirus-mediated suicide gene therapy for prostate cancer.

PURPOSE: Neoadjuvant gene therapy potentially improves the outcome of primary treatment of prostate cancer by radical prostatectomy in patients with high risk of recurrence. We conducted a Phase I escalating dose study with a replication-defective adenovirus expressing the herpes simplex virus-thymidine kinase gene (Adv-HSV-tk vector). The primary end point was toxicity, while the evaluation of the patients' cellular and humoral immune responses served as a secondary endpoint. MATERIAL AND METHODS: The Adv-HSV-tk vector was injected into the prostate in two doses (2x10(10) to 2x10(11) viral particles), followed by ganciclovir twice daily for 14 days and retropubic radical prostatectomy on day 21. Adenovirus-specific neutralizing, IgG and IgA antibodies were evaluated. Peripheral blood mononuclear cells (PBMC) were stimulated by Adv-HSV-tk and analysed for IFN-gamma production and 3H-thymidine incorporation. Prostate specimens were immunostained for B (CD20+) and for T (CD3+) lymphocytes. RESULTS: Toxicity was minor in all 8 patients treated. In the prostate, no virus related cytopathic effect could be observed. Dose-dependent infiltration of T and B lymphocytes in the whole prostate and in tumor areas was observed. Boosting of adenovirus-specific antibody responses was observed in 7 patients, and an increased adenovirus-specific PBMC proliferation and IFN-gamma production was seen after Adv-HSV-tk stimulation. CONCLUSION: Neo-adjuvant adenovirus-mediated cytotoxic gene therapy prior to prostatectomy for prostate cancer is feasible and safe in an outpatient setting for intraprostatic vector doses up to 2x10(11) viral particles. Activation of the immune system was observed. Application of higher vector doses may be considered.

Prostate specific membrane antigen (PSMA) is a tissue-specific target for adenoviral transduction of prostate cancer in vitro.

BACKGROUND: Adenovirus binds to the coxsackievirus and adenovirus receptor (CAR) as a first step in the process of cellular infection. This dependence on CAR potentially limits the use of adenovirus in gene therapy, since CAR is expressed in many tissues of the body, and expression of CAR may be low or lost upon progression of certain tumors. These limitations may be overcome by transductional targeting of adenovirus towards other cell surface molecules. We have evaluated the pantumoral epithelial cell adhesion molecule (EpCAM) and prostate specific membrane antigen (PSMA) as possible targets for adenoviral transduction of prostate cancer cells. METHODS: Bispecific antibodies, constructed as conjugates between an anti-adenovirus fiber knob Fab' fragment and anti-EpCAM or anti-PSMA monoclonal antibodies, were incubated with an eGFP-expressing adenovirus to retarget this vector. A cell panel, that includes two prostate cancer cell lines and four non-prostate control lines, were infected with serial dilutions of the retargeted vector and specificity of infection was determined. RESULTS: Receptor-specific transduction was obtained for both EpCAM and PSMA. PSMA-retargeting was shown to be selective for the prostate cancer cell lines. CONCLUSIONS: PSMA serves as a tissue-specific target for adenoviral vectors and may be applicable for gene therapeutical treatment of prostate cancer.

Validation of transrectal ultrasonographic volumetry for orthotopic prostate tumours in mice.

Orthotopic human prostate tumour models in athymic nude mice are regarded as being most suitable for fundamental and pre-clinical research on prostate cancer. The anatomic localization of the tumour in the pelvis, however, provides little possibility for monitoring tumour growth or regression. To assess time-related changes in orthotopic tumour volume, we applied transrectal ultrasonography (TRUS) to the murine prostate. This technique has the advantages of allowing accurate monitoring of tumours during therapeutic manipulations and a reduction of animal use due to a reduction of sacrificing endpoints. To validate the TRUS method, the mouse prostate reconstitution model, RM-9, and the prostate-specific antigen (PSA) producing human prostate cancer xenograft PC-346 were used. Volumetric calliper measurements were performed with a 30 MHz ultrasound probe designed for intra-arterial use in humans. Tumour weight, determined at various time-points, was found to be closely related to actual tumour weight (R = 0.99) and, in the PC-346 model, to the level of PSA in the plasma. Furthermore, the interobserver variation for TRUS was low for tumours above 50 mg. Thus, TRUS for murine prostate tumours proves to be an accurate, reproducible and sensitive method.

Bioavailability of lab-contaminated and native polycyclic aromatic hydrocarbons to the amphipod Corophium volutator relates to chemical desorption.

In the present study, the relationship between bioavailability of polycyclic aromatic hydrocarbons (PAHs) to benthic amphipods and the PAH desorption kinetics was examined. To that end, field-contaminated sediment was treated in three different ways. One subsample had no addition of PAHs and contained native PAHs only. To a second subsample, six PAHs (phenanthrene, fluoranthene, anthracene, pyrene, benzo[b]fluoranthene, and benzo[k]fluoranthene) were added in the laboratory. Two of the PAHs were added at higher concentrations to a third subsample, serving as a control for concentration-dependent uptake. Marine amphipods (Corophium volutator) were exposed to the three subsamples for a maximum of 25 d and were subsequently analyzed. Desorption kinetics were determined for both the lab-contaminated and the native PAHs. The biota-to-sediment accumulation factor (BSAF) values of the individual native and lab-contaminated PAHs correlated well with the rapidly desorbing fraction (R2 = 0.76). The BSAFs were 1.4 to 3.3 higher for the lab-contaminated PAHs compared with the native PAHs, while the difference between the rapidly desorbing fractions was a factor of 1.1 to 1.8. The BSAFs of the lab-contaminated PAHs in the second and third subsample were equal, indicating concentration-independent accumulation. The results suggest that lab-contaminated PAHs are more available to amphipods than native PAHs and that differences in bioavailability of lab-contaminated and native PAHs to marine amphipods are related to differences in desorption behavior.

Transrectal ultrasound for monitoring murine orthotopic prostate tumor.

BACKGROUND: The mouse orthotopic prostate tumor model has been recognized as an ideal preclinical animal model simulating the anatomical and biological milieu of the prostate. In comparison with the subcutaneous tumor model, the only disadvantage of this model is the difficulty of chronological tumor growth monitoring. We have applied recent endoluminal ultrasound technology, transrectal ultrasonography (TRUS), to the monitoring of mouse orthotopic prostate tumors. METHODS: A 6 Fr. 20 MHz catheter-based radial scan probe was used and TRUS was performed without any prior preparation including anesthesia. Orthotopic tumors were initiated by inoculation of 5000 RM-9 cells into the dorsal prostate of 12-week-old C57BL/6 male mice. The tumor growth was monitored by TRUS from day 3 to day 21. In addition, TRUS was performed to detect tumor growth suppression after intraperitoneal administration of cis-diamminedichloroplatinum (CDDP). RESULTS: By ultrasound, tumors became detectable 7 days after tumor cell inoculation. TRUS images were clear and parallel to actual tumor growth. The tumor volume (X) calculated by TRUS correlated significantly with the actual tumor weight (Y) measured at autopsy; Y = 101.653 + 1.174X (R = 0.930, P < 0.001). Similarly, tumor growth suppression induced by CDDP was clearly detected by TRUS with reasonable accuracy. CONCLUSIONS: A high resolution TRUS allows simple and reliable monitoring of in situ tumor growth and growth suppression, making the mouse orthotopic prostate tumor model more efficient.

Slow desorption of PCBs and chlorobenzenes from soils and sediments: relations with sorbent and sorbate characteristics.

The kinetics of slow desorption were studied for four soils and four sediments with widely varying characteristics [organic carbon (OC) content 0.5-50%, organic matter (OM) aromatic content (7-37%)] for three chlorobenzenes and five polychlorinated biphenyls (PCBs). Slowly and very slowly desorbing fractions ranged from 1 to 50% (slow) and 3 to 40% (very slow) of the total amount sorbed, and were observed for all compounds and all soils and sediments. In spite of the wide variations in sorbate K(OW) (factor 1000) and sorbent characteristics, the rate constants of slow (k(slow), around 10(-3) h(-1)) and very slow (k(very slow), 10(-5)-10(-4) h(-1)) desorption appeared to be rather constant among the sorbates and sorbents (both within a factor of 5). There was a good correlation (r(2) above 0.9) between the distribution over the slow, very slow and rapid sediment fractions and log K(OC), indicating that sorbate hydrophobicity may be important for this distribution. No correlation could be found between sorbent characteristics [OC, N, and O in the organic matter, polarity index C/(N+O), OC aromaticity as determined by CP-MAS (13)C-NMR] and slow desorption parameters (slowly/very slowly desorbing fractions+corresponding rate constants). The absence of (1) a correlation between k(slow) and k(very slow), respectively, and OC content, and (2) the narrow range of k(slow) and k(very slow) values, indicates that intra-OM diffusion is not the mechanism of slow or very slow desorption, because on the basis of this mechanism it would be expected that increasing OC content would lead to longer diffusion pathlengths and, consequently, to smaller rate constants. In addition, it was tested whether differential scanning calorimetry would reveal a glass transition in the soils/sediments. In spite of the sensitivity of the equipment used (changes in heat flow in the micro-Watt range were measurable), a glass transition was not observed. This means that activation enthalpies of slow desorption can be calculated from desorption measurements at various temperatures. In the present study these values ranged from 60 to 100 kJ/mol among the various soils and sediments studied.

Bioavailability in soil or sediment: exposure of different organisms and approaches to study it.

Soil and benthic organisms may be exposed to contaminants via different routes: (pore) water, soil or sediment, and food. Depuration of the contaminant from the organisms may take place via the same routes and, additionally, via biotransformation, reproduction, etc. Whereas uptake from and depuration to water can be predicted well, predictions for soil or sediment are less accurate. One of the reasons may be the reduced bioavailability of the contaminant in the soil or sediment. In biomimetic approaches, such as solid phase micro-extraction (SPME) or measurements with C18-discs, the freely dissolved concentration in the (pore) water is determined. The SPME-fiber or C18-disc may serve as a surrogate organism, but sometimes underestimates, and sometimes overestimates bioavailability. The soil (or sediment) availability ratio (SARA) method, that uses organisms to study the uptake of freshly added and 'aged' chemicals, is proposed to study the magnitude of the reduction in bioavailability. SARA also includes the organism-specific exposure and depuration routes.

Alternative splicing of follicle-stimulating hormone receptor pre-mRNA: cloning and characterization of two alternatively spliced mRNA transcripts.

Glycoprotein hormone receptors contain a large extracellular domain that is encoded by multiple exons, facilitating the possibility of expressing alternatively spliced transcripts. We have cloned two new splice variants of the rat follicle-stimulating hormone (FSH) receptor gene: FSH-R1 and FSH-R2. The splice variant FSH-R1 differs from the full-length FSH receptor mRNA by the inclusion of a small extra exon between exons 9 and 10. FSH-R2 lacks the first three base pairs of exon 4, contains an extra exon between exons 4 and 5, and has an extended 3'-untranslated region. According to the predicted open reading frames, both mRNAs encode truncated FSH receptor proteins, consisting of the entire extracellular domain (FSH-R1) or the amino-terminal half of the extracellular domain (FSH-R2), and are expressed at a low level in testes and ovaries. The levels of expression of the FSH-R1 and FSH-R2 mRNAs in the gonads show a constant ratio to the expression level of the full-length FSH receptor mRNA. Furthermore, in vitro co-expression of either one of the truncated proteins with the full-length FSH receptor in COS1 cells did not affect signal transduction through the full-length FSH receptor. The absence of a function of the truncated FSH receptors in FSH signal transduction in vitro, and the lack of differential regulation of the alternative transcripts, indicate that there is no clear function for alternative splicing of the FSH receptor pre-mRNA in the postnatal testis and the cycling adult ovary.