Rapid identification of lettuce seed germination mutants by bulked segregant analysis and whole genome sequencing.

Lettuce (Lactuca sativa) seeds exhibit thermoinhibition, or failure to complete germination when imbibed at warm temperatures. Chemical mutagenesis was employed to develop lettuce lines that exhibit germination thermotolerance. Two independent thermotolerant lettuce seed mutant lines, TG01 and TG10, were generated through ethyl methanesulfonate mutagenesis. Genetic and physiological analyses indicated that these two mutations were allelic and recessive. To identify the causal gene(s), we applied bulked segregant analysis by whole genome sequencing. For each mutant, bulked DNA samples of segregating thermotolerant (mutant) seeds were sequenced and analyzed for homozygous single-nucleotide polymorphisms. Two independent candidate mutations were identified at different physical positions in the zeaxanthin epoxidase gene (ABSCISIC ACID DEFICIENT 1/ZEAXANTHIN EPOXIDASE, or ABA1/ZEP) in TG01 and TG10. The mutation in TG01 caused an amino acid replacement, whereas the mutation in TG10 resulted in alternative mRNA splicing. Endogenous abscisic acid contents were reduced in both mutants, and expression of the ABA1 gene from wild-type lettuce under its own promoter fully complemented the TG01 mutant. Conventional genetic mapping confirmed that the causal mutations were located near the ZEP/ABA1 gene, but the bulked segregant whole genome sequencing approach more efficiently identified the specific gene responsible for the phenotype.

Mutation analysis of the LH receptor gene in Leydig cell adenoma and hyperplasia and functional and biochemical studies of activating mutations of the LH receptor gene.

CONTEXT: Germline and somatic activating mutations in the LH receptor (LHR) gene have been reported. OBJECTIVE: Our objective was to perform mutation analysis of the LHR gene of patients with Leydig cell adenoma or hyperplasia. Functional studies were conducted to compare the D578H-LHR mutant with the wild-type (WT)-LHR and the D578G-LHR mutant, a classic cause of testotoxicosis. The three main signal transduction pathways in which LHR is involved were studied. PATIENTS: We describe eight male patients with gonadotropin-independent precocious puberty due to Leydig cell adenoma or hyperplasia. RESULTS: The D578H-LHR mutation was found in the adenoma or nodule with hyperplasia in all but two patients. D578H-LHR displayed a constitutively increased but noninducible production of cAMP, led to a very high production of inositol phosphates, and induced a slight phosphorylation of p44/42 MAPK in the absence of human chorionic gonadotropin. The D578G-LHR showed a response intermediate between WT-LHR and the D578H-LHR. Subcellular localization studies showed that the WT-LHR was almost exclusively located at the cell membrane, whereas the D578H-LHR showed signs of internalization. D578H-LHR was the only receptor to colocalize with early endosomes in the absence of human chorionic gonadotropin. CONCLUSIONS: Although several LHR mutations have been reported in testotoxicosis, the D578H-LHR mutation, which has been found only as a somatic mutation, appears up until now to be specifically responsible for Leydig cell adenomas. This is reflected by the different activation of the signal transduction pathways, when compared with the WT-LHR or D578G-LHR, which may explain the tumorigenesis in the D578H mutant.

Unsaturated fatty acids prevent desensitization of the human growth hormone secretagogue receptor by blocking its internalization.

The composition of the plasma membrane affects the responsiveness of cells to metabolically important hormones such as insulin and vasoactive intestinal peptide. Ghrelin is a metabolically regulated hormone that activates the G protein-coupled receptor GH secretagogue receptor type 1a (GHSR) not only in the pituitary gland but also in peripheral tissues such as the pancreas, stomach, and T cells in the circulation. We have investigated the effects of lipids and altered plasma membrane composition on GHSR activation. Oligounsaturated fatty acids (OFAs) disrupt the structure of membranes and make them more fluid. Prolonged (96 h), but not acute, treatment of the GHSR cells with the 18C OFAs oleic and linoleic acid caused a significant increase in sensitivity of the receptor to ghrelin (EC(50) reduced by a factor of 2.4 and 2.9 at 60 and 120 microM OFAs, respectively). OFAs were found to block the inhibitory effects of ghrelin pretreatment on subsequent ghrelin responsiveness, suggesting that OFAs suppress desensitization of GHSR. Radioligand displacement studies did not show a significant shift in receptor binding after incubation with OFAs. However, it was found that OFA treatment suppressed GHSR internalization, likely explaining OFA-induced refractoriness to ligand-induced desensitization. The involvement of lipid rafts in this process was indicated by the altered responsiveness of GHSR under conditions that alter membrane cholesterol. In conclusion, our findings demonstrate the importance of membrane composition for GHSR activation and desensitization and indicate at least part of the mechanism through which OFAs and cholesterol could affect ghrelin's activity in vivo.

LH receptor gene expression is essentially absent in breast tumor tissue: implications for treatment.

Worldwide, breast cancer is the most frequently occurring malignancy in women. Early age at full term pregnancy has a protective effect against breast cancer. Evidence coming from a rat breast cancer model suggests a possible role for the pregnancy hormone hCG, a ligand of the LH receptor, as a mediator for this effect. In a previous study, we found that a common polymorphism in the LH receptor associates with tumor progression in premenopausal breast cancer patients, as carriers of the variant receptor showed a shorter disease free survival compared to non-carriers. How hCG and its receptor exert their effects on breast cancer, however, is unclear. One possibility is that these effects take place through LH receptors present in the ovaries, thereby influencing steroid hormone production. Another possibility is that the effects take place through LH receptors present in breast tumor cells themselves, as some studies have detected the receptor in both normal and neoplastic breast tissues and in breast cancer cell lines. To investigate whether a direct effect of LH signaling in breast cancer is likely, we measured LH receptor mRNA expression levels in 1551 breast tumors and 42 different human breast cancer cell lines using a qRT-PCR with a wide dynamic range. In addition, associations between LH receptor expression and clinico-pathologic factors were investigated. Assay validation showed that as little as ?10 copies per reaction volume of LH receptor cDNA could still be detected by our assay. We show that LH receptors are undetectable in 62% of breast tumor samples and 41 of 42 breast cancer cell lines. For the remaining samples we found expression levels to be very low. Although low, expression of the LH receptor appears to be associated with normal breast cells, favorable tumor characteristics and low tumor percentage. Since expression of the LH receptor in breast cancer cells is very low, it almost excludes the possibility of direct signaling effects. We therefore conclude that signaling effects of the LH receptor on breast cancer most likely take place by an indirect pathway through the ovaries.

Asp330 and Tyr331 in the C-terminal cysteine-rich region of the luteinizing hormone receptor are key residues in hormone-induced receptor activation.

The luteinizing hormone (LH) receptor plays an essential role in male and female gonadal function. Together with the follicle-stimulating hormone (FSH) and thyroid stimulating hormone (TSH) receptors, the LH receptor forms the family of glycoprotein hormone receptors. All glycoprotein hormone receptors share a common modular topography, with an N-terminal extracellular ligand binding domain and a C-terminal seven-transmembrane transduction domain. The ligand binding domain consists of 9 leucine-rich repeats, flanked by N- and C-terminal cysteine-rich regions. Recently, crystal structures have been published of the extracellular domains of the FSH and TSH receptors. However, the C-terminal cysteine-rich region (CCR), also referred to as the "hinge region," was not included in these structures. Both structure and function of the CCR therefore remain unknown. In this study we set out to characterize important domains within the CCR of the LH receptor. First, we mutated all cysteines and combinations of cysteines in the CCR to identify the most probable disulfide bridges. Second, we exchanged large parts of the LH receptor CCR by its FSH receptor counterparts, and characterized the mutant receptors in transiently transfected HEK 293 cells. We zoomed in on important regions by focused exchange and deletion mutagenesis followed by alanine scanning. Mutations in the CCR specifically decreased the potencies of LH and hCG, because the potency of the low molecular weight agonist Org 41841 was unaffected. Using this unbiased approach, we identified Asp(330) and Tyr(331) as key amino acids in LH/hCG mediated signaling.

Polymorphic variations in exon 10 of the luteinizing hormone receptor: functional consequences and associations with breast cancer.

Polymorphic variation of the LHR gene may affect receptor function and accordingly may influence ovarian steroid hormone action, including steroid hormone-dependent clinical outcome. The functional effects of two single nucleotide polymorphisms (SNPs), i.e. LHR 291Asn/Ser (rs12470652) and 312Ser/Asn (rs2293275) in the biologically interesting exon 10 of the LHR gene are described. Furthermore, ethnic diversity in allele frequencies and genotype distributions of both SNPs was determined. In addition associations with breast cancer were studied in 751 breast cancer patients. In vitro transfection studies revealed altered glycosylation status and increased receptor sensitivity for the 291Ser LHR variant. No functional consequences were observed for the 312SerAsn LHR SNP. The LHR 312Asn allele was slightly more often present in two independent breast cancer patient cohorts as compared to controls (OR=1.15; p=0.03 and 1.26; p=0.001, respectively). In conclusion, although functional changes of the LHR 291Ser candidate allele were observed, no associations with breast cancer were found, while the LHR 312Asn allele can be regarded as a weak breast cancer risk allele.

A common polymorphism renders the luteinizing hormone receptor protein more active by improving signal peptide function and predicts adverse outcome in breast cancer patients.

CONTEXT: Epidemiological and animal studies indicate a carcinogenic role of estrogens in breast tissue. The pituitary gonadotropin LH is an important regulator of estrogen production in premenopausal women, whereas even in women after menopause, 10-25% of ovarian steroid hormone production is LH dependent. OBJECTIVE: We hypothesized that an LH receptor (LHR) gene variant may affect LHR function and thereby influence disease outcome in breast cancer patients. DESIGN: The association of a polymorphic CTCCAG (Leu-Gln) insertion (insLQ), in the signal peptide encoded by exon 1 of the LHR gene with breast cancer risk, (disease-free) survival, and clinicopathological features was studied in a large cohort of 751 breast cancer patients with complete follow-up. Functional analysis of the insLQ-LHR and non-LQ-LHR (no LQ insertion) was carried out using transfection studies. RESULTS: We found a significant association between the insLQ-LHR and a shorter disease-free survival (hazard ratio, 1.34; confidence interval, 1.11-1.63; P = 0.003). The mechanism of the effect of insLQ on LHR function involves increased receptor sensitivity (insLQ-LHR has a 1.9 times lower EC(50) than non-LQ-LHR; P = 0.02) and plasma membrane expression (insLQ-LHR has 1.4 times higher B(max); P = 0.0006) rendering the insLQ-LHR allele more active. CONCLUSIONS: The insLQ polymorphism increases LHR activity, thereby shortening breast cancer disease-free survival, probably by increasing estrogen exposure in female carriers.

Insight into mutation-induced activation of the luteinizing hormone receptor: molecular simulations predict the functional behavior of engineered mutants at M398.

In this study, molecular simulations have been combined with site-directed mutagenesis experiments to explore M398(2.43), a LH (lutropin) receptor (LHR) site in helix 2 susceptible to spontaneous activating mutations, and to develop a computational tool for predicting the functionality (i.e. active or nonactive) of LHR mutants.Site-directed mutagenesis experiments engineered 15 different substitutions for M389(2.43), which resulted in variable levels of constitutive activity, inversely correlated with the size of the replacing amino acid. This inverse correlation is suggested to be mediated by I460(3.46), M571(6.37), and Y623(7.53), the tyrosine of the NPxxY motif. In fact, size reduction at position 398(2.43), which is concurrent with constitutive receptor activity, releases the van der Waals interactions found in the wild-type LHR between M398(2.43) and these three amino acids, resulting in structural modifications in the proximity to the E/DRY/W motif. An increment, above a threshold value, in the solvent accessibility of the cytosolic ends of helices 3 and 6 is the main structural feature shared by the active mutants of the LHR. This feature has been successfully used for predicting the functionality of the engineered mutants at M398(2.43), proving that molecular simulations can be useful for in silico screening of LHR mutants.

Luteinizing hormone (LH)-responsive Cushing's syndrome: the demonstration of LH receptor messenger ribonucleic acid in hyperplastic adrenal cells, which respond to chorionic gonadotropin and serotonin agonists in vitro.

In a substantial part of adrenal adenomas and hyperplasias from patients with Cushing's syndrome, cortisol production is controlled by the expression of aberrant hormone receptors on adrenocortical cells. We present in vivo and in vitro data of two patients with a LH-responsive Cushing's syndrome based on ACTH-independent bilateral adrenal hyperplasia. Patients 1 and 2 are women who presented with Cushing's syndrome and bilateral adrenal hyperplasia. Endocrine testing demonstrated absence of cortisol diurnal rhythm, insufficient cortisol suppression after 1 mg dexamethasone orally, and undetectable ACTH levels in both patients. Both patients were treated by laparoscopic biadrenalectomy. In in vivo testing, in patients 1 and 2, a profound cortisol rise was found after administration of GnRH [change in cortisol (Delta F), 118 and 106%, respectively], human CG (Delta F, 133 and 44%), LH (Delta F, 73 and 43%), ACTH (Delta F, 89 and 181%), and the 5-hydroxy-tryptamine receptor type 4 (5-HT(4)) agonists cisapride (Delta F, 141 and 148%) and metoclopramide (Delta F, 189 and 95%). In in vitro testing, adrenal cells from patient 2 responded, in a dose-dependent fashion, with cortisol production after exposure to human CG (Delta F, 45%), cisapride (Delta F, 68%), and metoclopramide (Delta F, 81%). ACTH induced cortisol production by cells from both patients (Delta F, 135 and 159%). In receptor studies, LH receptor mRNA was demonstrated in adrenal tissue of both patients but also in control adrenal tissue of two patients with persisting pituitary-dependent Cushing's syndrome treated by biadrenalectomy. In neither patient were mutations found in the ACTH receptor gene. LH-responsive Cushing's syndrome associated with bilateral adrenal hyperplasia may result from aberrant (or possibly increased) adrenal LH receptor expression. This variant is further characterized by adrenal responsiveness to 5-HT4 receptor agonists, possibly pointing to an interaction between LH and serotonin in the regulation of cortisol secretion. Despite the regulatory potential of LH and 5-HT4 receptor agonists on cortisol production in our patients, their adrenals seemed to be still sensitive to ACTH, both in vivo and in vitro.

Differential inhibition of 17alpha-hydroxylase and 17,20-lyase activities by three novel missense CYP17 mutations identified in patients with P450c17 deficiency.

The microsomal enzyme cytochrome P450c17 is an important regulator of steroidogenesis. The enzyme has two functions: 17alpha-hydroxylase and 17,20-lyase activities. These functions determine the ability of adrenal glands and gonads to synthesize 17alpha-hydroxylated glucocorticoids (17alpha-hydroxylase activity) and/or sex steroids (17,20-lyase activity). Both enzyme functions depend on correct steroid binding, but it was recently shown that isolated lyase deficiency can also be caused by mutations located in the redox partner interaction domain. In this article we present the clinical history and molecular analysis of two patients with combined 17alpha-hydroxylase/17,20-lyase deficiency and four patients with isolated 17,20-lyase deficiency. In these six patients, four missense CYP17 mutations were identified. Two mutations were located in the steroid-binding domain (F114V and D116V), and the other two mutations were found in the redox partner interaction domain (R347C and R347H). We investigated the activity of these mutated proteins by transfection experiments in COS-1 cells using pregnenolone, progesterone, or their hydroxylated products as a substrate and measuring 17alpha-hydroxylase- and 17,20-lyase-dependent metabolites in the medium. The mutations in the steroid-binding domain (F114V and D116V) of P450c17 caused combined, complete (F114V), or partial (D116V) 17alpha-hydroxylase and 17,20-lyase deficiencies, whereas mutations in the redox partner interaction domain (R347C and R347H) displayed less severe 17alpha-hydroxylase deficiency, but complete 17,20-lyase deficiency. These findings are consistent with the clinical data and support the observation that the redox partner interaction domain is essential for normal 17,20-lyase function of P450c17.

LH receptor defects.

In this article the role of LH receptor gene mutations in patients with aberrant sex differentiation is discussed. In a dominant autosomal familial form of precocious puberty in boys (familial male-limited precocious puberty) LH receptor gene mutations have been identified. These single amino acid changes, mostly found in the sixth transmembrane helix and the third intracellular loop of the transmembrane domain of the LH receptor, cause constitutive activation of LH receptor protein without the hormone present, resulting in precocious production of testosterone by the testicular Leydig cells. The large number of activating LH receptor mutations have allowed more precise molecular modeling of the LH receptor protein. In a rare hereditary form of 46,XY male pseudohermaphroditism known as Leydig cell hypoplasia, LH receptor gene mutations have been identified that completely or partially inactivate the LH receptor protein. Large gene deletions cause complete absence of the LH receptor protein, whereas other, more subtle missense mutations prevent the receptor from assuming an active conformation.