Inferring biallelism of two FSH receptor mutations associated with spontaneous ovarian hyperstimulation syndrome by evaluating FSH, LH and HCG cross-activity.

RESEARCH QUESTION: What is the cumulative effect of two follicle-stimulating hormone receptor (FSHR) mutations in spontaneous ovarian hyperstimulation syndrome (sOHSS) pathogenesis? Are these mutations in the mono- or biallelic state? DESIGN: Two FSHR mutations were found in a pregnant patient affected by sOHSS with no predisposing conditions. While the p.Asn106His mutation is novel, the p.Ser128Tyr mutation has been associated with sOHSS previously. The patient's FSHR gene was analysed by Sanger sequencing, and FSHR cDNAs carrying a single or both point mutations were created by mutagenesis in vitro. cAMP activation by recombinant FSH, luteinizing hormone (LH), human chorionic gonadotropin (HCG) and thyroid-stimulating hormone (TSH) was evaluated in transfected HEK293 cells by bioluminescence resonance energy transfer. RESULTS: All mutations decreased the 50% effective concentration of FSH calculated for cAMP (P < 0.05, n = 6), resulting in two- to 10-fold lower ligand potency. TSH failed to induce an FSHR-mediated increase in intracellular cAMP, while LH was approximately four-fold more potent than HCG in p.Ser128Tyr FSHR-expressing HEK293 cells despite lower cAMP plateau levels (P < 0.05, n = 5). The p.Ser128Tyr FSHR mutation was found to be responsible for an LH-/HCG-induced increase in cAMP when it was in the biallelic heterozygous state with p.Asn106His, but no increase in cAMP was induced in the monoallelic state. CONCLUSION: In-vitro data support that, in pregnant patients with sOHSS, the two FSHR mutations have an opposing effect on the pathogenesis of sOHSS and are in the biallelic heterozygous form, allowing HCG to induce a p.Ser128Tyr FSHR-mediated increase in cAMP.

FSH-receptor Ala307Thr polymorphism is associated to polycystic ovary syndrome and to a higher responsiveness to exogenous FSH in Italian women.

OBJECTIVE: Herein we analyzed FSH-R polymorphism at position 307 aiming (a) to assess the prevalence of the three allelic variants (Ala307Ala, Ala307Thr and Thr307Thr) in relation to the type of ovary and (b) to clarify if the allelic variant could influence the responsiveness to exogenous FSH. STUDY DESIGN: We prospectively studied a group of 106 Italian women undergoing in vitro fertilization (IVF), among which 40 were subjects with polycystic ovary syndrome (PCOS) and 66 were normo-ovulatory women with a normal ovarian morphology at transvaginal ultrasound. DNA extraction, denaturing high-performance liquid chromatography (dHPLC) and DNA sequencing were used to detect the FSH-R 307 polymorphic genotype and the whole exon 10 was analyzed. RESULTS: The heterozygote variant Ala307Thr was significantly more frequent than the homozygote variants in women with PCOS, whereas in normo-ovulatory women with normal ovary the three allelic variants had a comparable prevalence. Women bearing the Ala307Thr variant showed a higher ovarian responsiveness to exogenous FSH than normo-ovulatory subjects. CONCLUSIONS: The heterozygote FSH-R polymorphism Ala307Thr is significantly more frequent in women with PCOS than in normo-ovulatory subjects and is more frequently associated with a higher ovarian responsiveness to exogenous FSH.

Application of pyrosequencing to the identification of sequence variations in the cystic fibrosis transmembrane conductance regulator gene.

BACKGROUND: A high number of mutations associated with cystic fibrosis have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, most of which are rare and, therefore, hamper extensive molecular diagnosis. In couples undergoing prenatal diagnosis where no mutation is found in one or both partners, additional analysis of intragenic polymorphisms may allow for the identification of fetal alleles associated with cystic fibrosis. METHODS: We developed novel, rapid and accurate assays for CFTR genotype determination using pyrosequencing technology; a simple, automated and reliable technique with low cost. RESULTS: Assays were optimized for the identification of the seven most frequent CFTR mutations (p.DeltaF508, p.N1303K, p.G542X, c.2183AA>G, c.1717-1G>A, p.W1282X, p.R1162X) in the Italian population and two common intragenic polymorphisms (rs213950 and rs1800136). Blind validation on 15 known control samples, typed for each sequence variation, allowed correct identification of all 135 genotypes. CONCLUSIONS: We demonstrated that this procedure is highly specific for the identification of individual CFTR sequence variations associated with cystic fibrosis, allowing both population screening and prenatal diagnosis.

Mutations and polymorphisms of the FSH receptor (FSHR) gene: clinical implications in female fecundity and molecular biology of FSHR protein and gene.

UNLABELLED: The portion of chromosome 2, including the gene codifying the receptor of FSH (FSHR gene), can display point mutations that cause variations in the amino acid sequence of the receptor protein (FSHR protein). Some of these structural changes affect the receptor functional properties that may be enhanced (activating mutations) or impaired (inactivating mutations). Activating mutations confer to FSHR a higher responsiveness to FSH, making it constitutively active even in the absence of the ligand, or render it able to nonspecifically respond to other tropic hormones (e.g., TSH). Inactivating mutations reduce the receptor's function up to a total block, altering either the formation of the receptor-ligand complex, or FSH signal transduction. FSHR inactivating mutations may cause primary or secondary amenorrhea, infertility, and premature ovarian failure (POF), whereas activating mutations can predispose to ovarian hyperstimulation syndrome (OHSS) as a consequence of exogenous FSH administration, or with a spontaneous onset. Beside point mutations, FSHR gene polymorphisms at specific sites (e.g., codons 307 and 680) may influence FSHR protein responsiveness to exogenous FSH, and finally affect the effectiveness of in vitro fertilization (IVF) treatment as well as the likelihood of developing a severe OHSS as a consequence of superovulation. This review summarizes the current knowledge about the FSHR gene mutations and polymorphisms, illustrating in the first part their clinical consequences for female reproductive function. In the second part, it describes the techniques to study the FSHR gene sequence, and gives more details about the molecular biology of FSHR protein, of FSHR gene and its mutations. TARGET AUDIENCE: Obstetricians & Gynecologists, Family Physicians. LEARNING OBJECTIVES: After reading this article, the reader should be able to list clinical disorders related to mutations in the follicle stimulating hormone receptor (FSHR) gene, explain the principles behind the study of FSHR mutations, and state possible future applications of knowledge of mutations in the FSHR gene in reproductive medicine.

Integrated strategy for fast and automated molecular characterization of genes involved in craniosynostosis.

BACKGROUND: Craniosynostosis, the premature fusion of 1 or more sutures of the skull, is a common congenital defect, with a prevalence of 1 in 2500 live births. Untreated progressive craniosynostosis leads to inhibition of brain growth and increased intracranial and intraorbital pressure. The heterogeneity of clinical phenotypes and the overlap of the various associated syndromes render the correct diagnosis of the different craniosynostoses particularly difficult. METHODS: To identify 10 common mutations in the genes for fibroblast growth factor receptors 2 and 3 (FGFR2 and FGFR3), we developed a microelectronic microchip assay that exploited the PCR multiplexing format and coupled it with serial addressing and probe hybridization on the same pad. For the molecular characterization of patients who tested negative in the microchip screening, we also developed conditions for denaturing HPLC (DHPLC) analysis of the most mutated regions of FGFR2 and FGFR3 and the entire coding region of the TWIST1 gene. RESULTS: In our cohort of 159 patients with various craniosynostosis syndromes, mutations were found in 100% of patients with Apert syndrome, 83.3% with Pfeiffer syndrome, 72.7% with Crouzon syndrome, 50.0% with Saethre-Chotzen syndrome, 27.7% with plagiocephaly, 31.8% with brachicephaly, 20% of complex cases, and 6.9% of mixed cases. No mutations were found in syndromic cases. CONCLUSIONS: The combined microchip-DHPLC strategy allows rapid and specific molecular diagnosis of craniosynostosis and is an effective tool for the medical and surgical management of these common congenital anomalies in a newborn or an infant with a developmental defect of the cranial vault.