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

bial-lelism

Human FSH, LH and TSH are dimeric, pituitary glycoprotein hormones sharing a common α and having a specific β-subunit (Alevizaki and Huhtaniemi, 2002).hCG is the placental gonadotropin which binds the LH receptor (LHCGR) and may cross-interact with the TSH receptor (TSHR) (De Leener et al., 2008).Similarly, TSH binding to LHCGR was also demonstrated (Hidaka et al., 1993), suggesting the presence, at the receptor level, of a relatively low number of key-residues responsible for the ligand specificity (G.Smits et al., 2003).Within the physiological hormone range, β-subunit provides receptor-specific binding to the extracellular leucine-rich domain of their structurally similar receptors (Moyle et al., 2004), avoiding promiscuous activation of cAMP/PKA and other intracellular signalling cascades (Costagliola et al., 2005).
Several cases of sOHSS have been reported, and reviewed (Lussiana et al., 2008;Meduri et al., 2008), in women carrying a mutated form of FSH receptor (FSHR).These mutations may change the sensitivity to FSH or induce loss of binding specificity, leading to cross-interaction between the receptor and other glycoprotein hormones, mainly TSH and hCG (Montanelli et al., 2004).
In this study, we evaluated single and additive effects of two missense FSHR mutations carried by a sOHSS patient, measuring the cAMP production induced by treatment of a receptoroverexpressing cell line by recombinant FSH, LH, hCG and TSH, in vitro.However, DNA

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A N U S C R I P T 4 4 sequencing did not reveal whether these heterozygous mutations are individually located on two different (biallelic) or on the same allele (monoallelic) (figure 1).At the protein level, mutations fall within the extracellular domain of FSHR and result in the asparagine to histidine amino acid change at position 106 of the protein chain (p.Asn106His), and in the serine to tyrosine change at position 128 (p.Ser128Tyr).While the p.Asn106His FSHR mutation is novel, the p.Ser128Tyr was previously associated with sOHSS and displayed hCG-, but not TSH-dependent cAMP increase in vitro and lack of constitutive activity (De Leener et al., 2008).However, the effect of LH was not tested.Herein we administrated glycoproteins in molar terms, instead of IU/ml, providing a precise, quantitative comparison between the molecules and their effects mediated by mutant FSHRs.

sOHSS patient
A patient with a clinical picture of sOHSS came to our observation.She was a 30 years-old woman who asked for a legal, voluntary abortion at 11 weeks of pregnancy.She was admitted to the hospital with severe abdominal pain and distension, dyspnea, oliguria.The blood tests revealed increased haematocrit, increased serum creatinine, hyponatremia with increased potassium levels.
TSH circulating levels were normal.The abdominal ultrasound showed severely enlarged ovaries (13 and 14 cm mean diameter) with multiple transonic cysts, and abundant ascites.Some pleural free fluid was also observed in the thorax radiography.The cytological examination of the abdominal fluid was negative for neoplastic cells.A brain magnetic resonance (MRI) was performed to exclude a pituitary adenoma.Blood hCG was monitored and it decreased normally after the abortion.The final diagnosis was severe sOHSS (Grade B according to previously published criteria) (Rizk and Aboulghar, 1991).Further ultrasound and hematological evaluations were scheduled every 15 days and documented the progressive reduction of ovarian volume and the complete disappearance of ascites with normalization of the blood exams.
PCR products were purified using the QIAquick ® PCR Purification Kit (Qiagen, Venlo, Netherlands) according to manufacturer's instructions.Sanger sequencing reactions were performed for 4 ng of each amplicon using the BigDye Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific).
The sequence reactions included 28 cycles consisting in 10 s at 96°C, 5 s at 50°C and 4 min at 60°C.Products obtained from the sequence reactions were purified with the DyeEx® 2.0 Spin Kit (Qiagen) and then analyzed on the ABI PRISM ® 3100-Avant Genetic Analyzer (Applied Biosystems).

Mutagenesis of FSHR cDNA
Starting from the previously described wild-type FSHR cDNA inserted in a pTracer vector (Thermo Fisher Scientific, Waltham, MA, USA) (Casarini et al., 2016) Mutant FSHR cDNAs inserted in the pTracer vector were amplified after thermal shock in supercompetent XL1-Blue Escherichia coli transformation (Agilent Technologies) following the manufacturer's instruction.Transformed bacteria grown over-night in pH=7.4Luria broth-agar plates (Sigma Aldrich), in the presence of 100 µg/ml ampicillin, at 37°C 8 h.Resistant clones were selected for growing over-night at 37°C and plasmids extracted by GenElute HP Endotoxin-Free Plasmid Maxiprep Kit (Sigma-Aldrich) following the procedure indicated by the provider.
Sequence reactions were performed in a total volume of 20 µl, including 3.5 pmol of forward and reverse primer, 200 ng of template, plasmid DNA, 2 µl BigDye ® Terminator v1.1 (Thermo Fisher Scientific).PCRs were performed by 40 cycles with 10 s at 96°C, 15 s at 50°C and 4 min at 60°C.
Products were sequenced by the ABI 3130 Genetic Analyzer (Applied Biosystems).
Expression of mutant and wild-type FSHR proteins in HEK293 cells were checked by Western blotting, using a previously validated method and reagents (Casarini et al., 2014).Briefly, lysates from 3x10 5 , 48-h transiently transfected cells were subjected to SDS-page electrophoresis and transferred on nitrocellulose membrane.Membranes were incubated over-night at 4°C with a rabbit anti-FSHR (Thermo Fisher Scientific) diluted 1:500 in tris-buffered saline (TBS) (Sigma-Aldrich).

Results
Patient's DNA and plasmid sequencing DNA sequencing showed that the patient carries a heterozygous mutation in exon 4 of the FSHR gene, leading to the p.Asn106His amino acid substitution at the protein level (figure 2, panel A).
Moreover, cytosine to adenine substitution, linked to the known p.Ser128Tyr mutation on exon 5 in heterozygosis, was also found (figure 2, panel B).
[FIGURE 2 A, B] Mutant FSHRs were produced by mutagenesis and checked by DNA sequencing (figure 3).In this case, two mutant FSHRs, carrying the single mutation each, were produced starting from the wildtype receptor (FSHR p.Asn106His; FSHR p.Ser128Tyr; figure 3, panel A and B, respectively).To evaluate the additive effect of both the mutations, an additional receptor was produced (FSHR p.Asn106His+p.Ser128Tyr; figure 3, panel C).

Evaluation of mutant FSHR expression in transfected HEK293 cells
Expression levels of the wild-type and mutant FSHR proteins were evaluated by Western blot analysis in transiently transfected HEK293 cells.Untransfected HEK293 and COS-7 cells served as a negative control, while positive control was human primary granulosa cell lysates and β-actin was the normalizer (figure 4).
[FIGURE 4] Wild-type and mutant FSHR cDNAs were similarly expressed, as a result of their insertion in front of the plasmid cytomegalovirus promoter.FSHR protein signal was detected at the level of granulosa cells lysate, while receptor expression was missing in untransfected HEK293 cells.values was performed.To this purpose, HEK293 cells were transfected using a cAMP biosensorexpressing plasmid and exposed to nM-µM concentrations of 8-br-cAMP.Data were plotted in a X-Y graph as mean±SD (n=8) and interpolated by non-linear regression (figure 5).

Analysis of FSH-, LH-, hCG-and TSH-induced cAMP production
Wild-type and mutant FSHR-overexpressing HEK293 were stimulated by pM-µM concentrations of recombinant glycoprotein hormones.cAMP was measured by BRET technique, expressed as mean±SD and indicated in molarity (figure 6) after extrapolation of the concentration from the standard curve (figure 6).50 µM forskolin was used to obtain the positive control.
[FIGURE 7] Since both the wild-type and the monoallelic p.Asn106His+p.Ser128Tyr FSHRs failed in mediating hCG-induced cAMP (figure 6), the effect in monoallelic heterozygous form, as a combination of these two conditions consisting in one wild-type FSHR and one FSHR allele carrying both the mutations, is reasonably expected to be the same (supplementary figure 1).

Discussion
In this study, we characterized single and additive effects of the missense FSHR mutations p.Asn106His and p.Ser128Tyr, that were found in a patient complaining of sOHSS.The functional characterization was carried out by treatment of transfected HEK293 cells, using recombinant gonadotropins and thyrotropin, and evaluating intracellular cAMP increase.While both the mutations appeared to be linked to decreased sensitivity of the receptor to the natural ligand FSH, the p.Ser128Tyr induced loss of specificity, resulting in LH-and hCG-induced cAMP increase.TSH did not induce significant effects, except at a relatively high concentration, 1 µM, which induced weak cAMP activation mediated by wild-type and p.Ser128Tyr mutant FSHR.LH and hCG crossreactivity linked to the p.Ser128Tyr mutation was dampened by the presence of the amino acid histidine at position 106, reflected in lower EC 50 and plateau cAMP levels.
The p.Ser128Tyr amino acid change was previously associated to sOHSS (De Leener et al., 2008).This mutation falls within the exon 5 of the FSHR gene and is linked to increased affinity and sensitivity to hCG, without conferring constitutive activity to the receptor or promiscuous activation by TSH.We found this mutation in a pregnant women affected by sOHSS during the first trimester of pregnancy, when hCG achieves physiologically maximal levels (Mishell and Davajan, 1966).Taken together, these data suggest that the p.Ser128Tyr change may not be causative of FSH-dependent sOHSS development during the fertile windows of women, outside pregnancy.
Interestingly, cAMP increase induced by 10-100 nM LH indicates cross-interaction with the p.Ser128Tyr mutant receptor, unlikely resulting in sOHSS risk during the midcycle LH peak, when the hormone serum concentration is about 10-100-fold lower and no significant FSHR expression occurs (Jeppesen et al., 2012).Most importantly, the LH results in about 4-fold lower EC 50 value than hCG, likely due to different binding affinity to the mutant receptor.Indeed, our results indicate that tyrosine at position 128 is more selective for LH than for the structurally similar hCG.Previous data demonstrated the non-equivalence of the two hormones, revealing that hCG is about 5-fold more potent than LH in inducing cAMP production in human primary granulosa cells (Casarini et al., 2012), mouse Leydig cells (Riccetti et al., 2017a) and transfected cell lines (Riccetti et al., 2017b).These features are likely due to ligand-specific tridimensional conformations of the hormone-receptor complex (Grzesik et al., 2015), rather than different binding affinity for the natural receptor, LHCGR.
The introduction of the amino acid histidine at position 106 of FSHR decreases binding affinity for the natural ligand, FSH, as well as LH, hCG and TSH cross-reactivity.These data indicate that asparagine at position 106 is important for the occurrence and specificity of FSH binding to its receptor.Moreover, these data suggest a lack of association of this novel FSHR mutation with sOHSS, since no results support hyper-responsiveness of the p.Asn106His mutant FSHR to endogenous glycoprotein hormones.On the contrary, this amino acid change may dampen the increased sensitivity to hCG, provided by the known p.Ser128Tyr FSHR mutation, when they are both carried by the same protein receptor but not in the biallelic heterozygous form.
In summary, our results suggest that, in our sOHSS patient, the two mutations occurred in biallelic heterozygous form, resulting in one allele with decreased sensitivity to glycoprotein hormones and one allele able to cross-interacts with hCG during pregnancy.These results provide an independent confirmation that p.Ser128Tyr FSHR induces hCG-induced sOHSS, while LH midcycle peak should not be a risk for developing the disease in women expressing the mutant receptor.Moreover, although asparagine and histidine share similar chemical properties, amino acid change at position 106 is crucial for proper FSHR functioning, suggesting that homozygous mutations falling within this region might be linked to infertility.Taken together, these findings reflect the individual-specific nature of the cell response to hormone stimulation and point out the relevance of proper pharmacogenetic approaches to be applied for clinical treatment.In this case, the evaluation of signaling activation using transfected cell line may provide data indicative of the hormonal treatment in vivo and a tool to be used for preliminary testing of the cell response associated to peculiar gonadotropin receptor genotypes.

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consent for genetic screening.
Signals were developed after treatment by ECL Advance Western Blotting Detection (Perkin Elmer Inc., Waltham, MA, USA), acquired using the VersaDoc system (BioRad Laboratories) and semiquantified by the Quantity-One software (BioRad Laboratories).Cell stimulations and Bioluminescence Resonance Energy Transfer (BRET) measurements48-h transiently transfected cells were washed and maintained in 40 µl/well of 37°C PBS and 1 mM HEPES, in the presence or in the absence of 1 pM-1 µM FSH, LH, hCG or TSH.Cells treated by 50 µM of the adenylyl cyclase activator forskolin (Sigma-Aldrich Corporation) served as positive controls.BRET measurements were performed upon addition of 10 µl/well of 5 µM coelenterazine H (Interchim, Montluçon, France), using the CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany).Donor and acceptor signals were detected at the wavelength of 480±20 and 540±20 nm, respectively, and represented as a ratio (BRET changes).In order to extrapolate real cAMP concentrations from BRET change values, in FSH-stimulated cells, a cAMP standard curve was generated.To this purpose, HEK293 cells were transfected using the cAMP biosensor-expressing plasmid and exposed 20 min to increasing concentrations (0-10 µM range) of the analog 8-bromoadenosine 3,5-cAMP (8-br-cAMP; Sigma-Aldrich Corporation).Under these conditions, endogenous cAMP was undetectable.BRET change values were plotted on a X-Y graph, where 8-br-cAMP concentrations were on the logarithmic X-axis, while BRET change on the Y-axis.Data were interpolated using a non-linear regression, which was used for extrapolation of real cAMP concentrations.StatisticsAnalysis of cAMP dose-response data were represented as BRET changes over basal.Data were represented as mean±standard deviation (SD) in a graph with logarithmic X-axis and interpolated by non-linear regression.The 50% effective concentration (EC 50 ) was calculated for each doseresponse curve.Departure from normality was evaluated by D'Agostino and Pearson test and comparisons between couples of EC 50 values were performed by Mann-Whitney's U-test.Multiple comparisons between dose-response curves were performed using the two-way ANOVA and A C C E P T E D M A N U S C R I P T 9 9 for extrapolating real cAMP values, expressed in molarity, from BRET change