JCEM-2015

Functional characterization of two naturally occurringmutations (Val514Ala and Ala575Val) in folliclestimulating hormone receptor

Swapna S. Desai,1 Swati K. Achrekar,1 Krupa A. Sahasrabuddhe,1

Parvin K. Meharji,1 Sadhana K. Desai,2 Vijay S. Mangoli,2 Smita D. Mahale1,3

1National Institute for Research in Reproductive Health, Jehangir Merwanji St, Parel, Mumbai- 400 012,India; 2Fertility Clinic and IVF Centre, 101/102 Shanti Niketan, V Gandhi Rd, Gamdevi, Mumbai- 400007, India

Context: Inactivating mutations have been reported in subjects with primary/secondary amenor-rhea, whereas, activating mutations are rare and seen only in women with ovarian hyper stimu-lation syndrome (OHSS). In the present study, we describe the functional characterization of thetwo mutations Val514Ala (novel mutation) and Ala575Val in FSHR identified in women with OHSSdeveloped during in vitro fertilization (IVF) and primary amenorrhea respectively.

Objective: To study the effect of mutations (514 and 575) on FSHR activity by in vitro functionalstudies.

Setting: Academic research institute and private IVF clinic.

Methods: The site-directed mutagenesis was carried out to generate the mutations at position 514and 575 in pSG5-FSHR construct. Stable cell lines expressing wild type or each of the mutantreceptor was generated using CHO cells. Functional characteristics of both the mutant receptorswere assessed by radio receptor assay and cAMP assay.

Results: The mutant receptor 514 showed increased cell surface expression as compared to wildtype (WT) receptor. Though the hormone binding characteristics were similar to WT receptor itssignaling activity was distinctly higher at lower dose of FSH as monitored by cAMP assay. On theother hand, the mutant receptor 575 showed lower cell surface expression and higher internalizedhormone-receptor complex. Additionally, dose dependent increase in the cAMP accumulation wasnot observed in case of this mutant as compared to WT.

Conclusion: OHSS and primary amenorrhea observed in the two affected woman respectively,could be attributed to the functional characteristics of respective mutant FSHR.

Follicle-stimulating hormone (FSH) belongs to the gly-coprotein hormone family which includes luteinizing

hormone (LH), chorionic gonadotropin (CG) and thyroidstimulating hormone (TSH). The role of FSH in regulatingthe gonadal function has been well documented (1). Itbrings about its physiological action by binding to specificFSH receptor (FSHR), which belongs to the super familyof glycoprotein hormone receptors (GPHRs). The mem-bers of GPHRs are transmembrane proteins of which

FSHR is predominantly expressed on the membrane ofgranulosa cells in the ovary (2). Upon binding to its ligand,FSHR facilitates multiple signaling pathways. In females,FSH-FSHR interaction triggers growth and maturation ofovarian follicle leading to steroidogenesis (3). Studies onthe targeted deletion of FSHR gene in female mice reportedcomplete arrest of folliculogenesis causing infertility (4).Interestingly, the gain-of-function mutation identified inmouse FSHR has clearly demonstrated prominent changes

ISSN Print 0021-972X ISSN Online 1945-7197Printed in U.S.A.Copyright © 2015 by the Endocrine SocietyReceived September 27, 2014. Accepted January 7, 2015.

Abbreviations:

O R I G I N A L A R T I C L E

doi: 10.1210/jc.2014-3662 J Clin Endocrinol Metab jcem.endojournals.org 1

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in ovarian function (5). Thus, FSHR is indispensable infemale fertility and any loss/gain of its function may leadto impaired folliculogenesis.

The first inactivating mutation Ala189Val in FSHR genewas reported from Finnish population in the subjects withovarian dysgenesis (ODG1) (6). Thereafter, very few in-activating mutations in FSHR gene have been reported (7).Most of these inactivating mutations have been observedto be defective in trafficking of the receptor to the cellsurface, leading to reduced expression of the receptor onthe cell membrane (8). Such studies have helped in estab-lishing a genotype-phenotype correlation and also con-tribute in understanding the significance of affected aminoacid in FSHR function (6, 9, 10).

On the other hand, in case of activating mutations, thegain of function of the receptor has been reported. So far,only two constitutively activating FSHR mutations inmale have been reported (11, 12). In females, very fewcases of spontaneous ovarian hyperstimulation syndrome(sOHSS) have been elucidated by identification of muta-tions in FSHR leading to the activation of the receptor(13). These mutations have been reported to cause loss ofligand binding specificity and respond to hCG resulting inhyper stimulation (13). Indeed OHSS is usually a compli-cation of gonadotropin stimulation during in vitro fertil-ization (IVF) protocol where it is known as iatrogenicOHSS (aOHSS); and very rarely it could be associatedwith spontaneous gestation. The cause of hyper respon-siveness of ovaries to gonadotropin stimulations duringIVF is still largely unknown and needs to be explored (14).

Our group has been working on the variants of FSHRgene and their significance in receptor function (13). To-wards this, we had screened for the FSHR mutations inwomen from Indian population undergoing infertilitytreatment. To our interest, we identified two mutations inFSHR in two different subjects manifesting phenotypicvariations. In the present study, we describe the functionalcharacterization of these two mutations to understand thesignificance of affected residues in receptor function.

Subjects and Methods

Identification of mutations in FSHR geneFor screening the FSHR gene variants, we had recruited two

distinct groups of females viz; (i) normogonadotropic womenundergoing IVF protocol (n � 50) and (ii) women with primaryand secondary amenorrhea (n � 86). The proven fertile healthyfemales (n � 100) were recruited as control group (15). In ourattempt to detect known/novel mutations in FSHR gene, theentire exonic region was screened as described earlier (15). Weidentified two mutations of which; one is a novel mutationVal514Ala presented in a subject undergoing IVF and the othermutation Ala575Val is described earlier (15). We further screened

additional subjects undergoing IVF (n � 100) recruited duringour previous study (16) to assess for the presence of both thesenovel FSHR mutations. The mutation at position 514 wasscreened by direct sequencing and mutation at position 575 wasscreened by RFLP technique as described earlier (15). All thesubjects were of Indian origin. The study was approved by theNational Institute for Research in Reproductive Health humanethics committee and informed consent was obtained from all thestudy subjects.

Phenotype of the subjects identified with themutation at position 514 and 575case 1. The subject with mutation at position 514 was a 27-year-old normogonadotropic female with normal karyotype (46,XX). She was undergoing FSH stimulation in an IVF clinic dueto cause of male as well as tubal factor. She was also presentedwith polycystic ovaries before undergoing IVF. Her serum FSH(3.2 mIU/ml) and LH (3.7 mIU/ml) levels on day 3 (basal) werewithin the normal range. Total amount of exogenous FSH ad-ministered during the IVF cycle was 3225 IU. Her estrogen levelsbefore and after hCG stimulation were 6284 pg/ml and 7111pg/ml respectively. Number of preovulatory follicles and oocytesretrieved were 20 and 29 respectively. The subject had developedaOHSS during FSH stimulation.

case 2. The mutation at position 575 was identified in a subjectwith primary amenorrhea with poor secondary organ develop-ment. No pubic and axillary hairs were seen. This subject was16-year-old female with normal karyotype (46, XX). Her serumFSH and LH levels were 76 and 35 mIU/ml respectively. She wastreated with estrogen and progestin for the period of ninemonths. However, her serum FSH levels after treatment was 56mIU/ml. Ultrasound findings after treatment revealed normaluterus, small ovaries with no follicles and thin endometrium(15).

MutagenesisThe mutations Val514Ala (Accession no. FJ211855) or

Ala575Val (Accession no. FJ211856) (15) was introduced intothe pSG5-hFSHR plasmid vector (17) by oligonucleotide medi-ated mutagenesis using Quick-change Site-directed MutagenesisKit (Stratagene, La Jolla, CA, USA). The full length FSHR cDNAsequence in both the mutant constructs was verified by directsequencing.

Generation of CHO cells expressing WT/mutantFSHR

For generation of stable cell line, the hFSHR expression con-structs (1 �g/well) (pSG5-hFSHR WT/mutants at position 514and 575) were transiently cotransfected along with the emptypcDNA3.1 vector (for G418 resistant marker) in 10:1 dilution inChinese hamster ovary (CHO) cells (1 � 105 cells/dish) in a 65mm dish using lipofectamine-plus transfection reagent accordingto the manufacturer’s instructions (Invitrogen, CA, USA) in se-rum-free DMEM-F12 medium. After 3 hours of incubation at 37C, medium containing FBS (5%) and neomycin analog G418(1000 �g/ml) was added. Cells were further cultured for 14 daystill distinct clones were formed. After 14 days, G418-resistantstable clones were selected (20 clones for each construct) andscreened for FSHR expression by Western blotting. The positive

2 Mutations in FSHR gene J Clin Endocrinol Metab


clone for each, WT and mutant constructs were then culturedand used for further functional characterization. The stable cellline transfected only with empty vector pcDNA3.1 was also gen-erated as described above and used as a negative control in all theexperiments.

Western blot analysisThe protocol was adapted from Desai et al (16) with minor

modifications. The stable cells expressing WT/mutant FSHR atposition 514 and 575 were lysed on ice in a lysis buffer (1%Igepal, 0.4% deoxycholate, 10 mM Tris pH 7.5, 6.6 mM EDTA,140 mM sodium chloride and containing protease inhibitors).Twenty �g of the respective lysates were then subjected to SDS-PAGE and Western blotting using Immobilon-P membranes(Millipore, Bedford, MA, USA).

Ligand binding studiesBinding of 125I-FSH to the WT/mutant FSHR was studied

using stable cells (2 � 105 cells/well) cultured in 24-well plates.The radio-iodination of hFSH (procured from National Hor-mone and Pituitary Programme, NIDDK, Bethesda, MD, USA)was carried out by the iodogen method (18). The radio receptorassay (RRA) and displacement assay were carried out as de-scribed earlier by Dupakuntla et al (19).

FSH induced signalingFSH-induced cAMP production was measured in CHO cells

expresing WT/FSHR mutants. Cells (2 � 105 cells/well) werestimulated with hFSH (doses ranging from 0 to 100 ng) for 1hour. In another set of experiment, cells expressing WT andmutant receptor 514 were stimulated with different doses ofhCG/TSH (10 and 100 ng/well) for 1 hour to assess for the ligandspecificity of the receptor. The cells were lysed with 0.1 M HCland subjected to centrifugation. Supernatants were diluted inassay buffer (1:1 dilution) and the cAMP levels were estimated byenzyme immunoassay (EIA) using a cAMP kit (Cayman Chem-ical Company, Ann Arbor, MI, USA), according to the manu-facturer’s instructions.

Confocal microscopyDirect immunofluorescence was performed on the stable cells

expressing WT/mutant FSHR. The protocol was adapted fromBeau et al (20) with minor modifications. The FSHR mAb106.105, was labeled with Alexa Fluor 568 using a commercialkit (Molecular Probes, Life Technologies, Carlsbad, CA, USA).For permeabilization, the cells were treated with 5% NP40 inPBS for 10 minutes prior to incubating with labeled antibody.Images were captured using an oil immersion 63X objective withNA 1.4 on a LSM510-Meta confocal system (Carl Zeiss, Jena,Germany).

Results

Mutations in the FSHRTwo mutations at Val514Ala (novel) and Ala575Val

were identified in the recruited subjects. None of theproven fertile control subjects (n � 100) and additionalsubjects (n � 100) undergoing IVF showed presence of anyof these two mutations, further providing evidence that itis not a common polymorphism. The first mutation atamino acid position 514 was identified in a subject un-dergoing gonadotropin stimulation and had developedaOHSS during IVF. This was observed to be a homozy-gous mutation leading to transition of thymidine to cyto-sine at nucleotide position 1540 (GT1540G to GCG) inExon 10 yielding a Val514Ala substitution in the Exo-loop2 (EL2) of the FSHR (Figure 1A). The second mutationwas observed to be a homozygous transition of cytosine tothymidine at nucleotide position 1723 (GC1723C to GTC)in Exon 10 of FSHR gene. This mutation was identified atposition 575 of FSHR in a woman with primary amenor-rhea which results in a substitution of Ala to Val in thesixth TMH of FSHR (15). Location of both these residues

Figure 1. A, Electropherogram of 5�- 3� strand showing sequences of control subject (upper panel) and homozygous mutation (lower panel)resulting in change of nucleotide T to C (1540 position) leading to Val514Ala substitution identified in an woman undergoing IVF (case 1). B,Schematic representation of FSHR showing the position of naturally occurring novel mutations at Val514Ala and Ala575Val.

doi: 10.1210/jc.2014-3662 jcem.endojournals.org 3


in FSHR has been shown (Figure 1B). It was interesting tonote that both these mutations are conserved across theGPHRs (FSHR, LHR and TSHR) as well as across differ-ent species (Figure 2).

Expression of FSHR in transfected cellsMature form of FSHR at around 80 kDa was observed

to be present in stable cells expressing either the WT ormutant receptors. The total protein expression was ob-served to be almost similar in all three cell lines expressingWT or mutant FSHR (Figure 3).

Effect of mutation on binding of FSH to FSHRThe CHO cells expressing WT/mutant FSHR were used

to study the binding of the 125I-FSH to receptor by RRAin the presence/absence of unlabeled FSH. The level ofsurface bound hormone was observed to be significantlyhigher in mutant receptor 514 as compared to WT recep-tor. However, internalized hormone-receptor complexwas lower as compared to WT FSHR, leading to signifi-cantly lower internalization index. In case of mutant re-ceptor 575, the level of bound hormone to the receptor onthe cell surface was significantly lower than WT FSHR.However, the internalization of the hormone-receptor

complex was evidently higher result-ing in significantly higher internal-ization index (Figure 4A, 4B). Fur-ther, the mutant receptors wereanalyzed for ligand binding charac-teristics by displacement assay. Thedisplacement curve between the WT(EC50 � 10.65 ng) and the mutantreceptors 514 (EC50 � 9.40 ng) and575 (EC50 � 9.74 ng) was observedto be parallel and possessed similarbinding kinetics towards FSH (Fig-ure 4C).

Effect of mutation on FSH induced signalingFSH induced signaling activity of the receptor was stud-

ied using stable cells expressing either WT or mutant re-ceptor. In case of WT FSHR, dose dependent increase inthe cAMP levels was observed. The basal cAMP levelswere not significantly higher in case of both the mutantreceptors as compared to WT. At the low dose (12.5 and25 ng) of FSH stimulation, the cells expressing mutantreceptor 514 showed higher accumulation of cAMP ascompared to WT receptor. However, the signaling activityremained almost similar on increasing the dose (50 and100 ng) of FSH (Figure 4D). To assess the ligand bindingspecificity of mutant receptor 514, cells were stimulatedwith hCG/TSH. The accumulation of cAMP upon stimu-lation with different doses of hCG/TSH (10 and 100 ng)was not observed. Similar results were observed in case ofWT FSHR (data not shown). In case of mutant receptor575, dose dependent increase in the cAMP was not ob-served upon FSH stimulation (Figure 4D).

Effect of mutation on trafficking of receptor on tothe cell surface

The surface localization of these cells was evaluatedusing immuno-fluorescence technique. In the nonperme-abilized condition, the surface expression was observed tobe higher in cells expressing mutant receptor 514 as com-pared to WT receptor. In case of mutant receptor 575, thesurface expression was weak. However, upon permeabi-lization, both the mutant receptor showed similar intra-cellular expression compared to WT FSHR (Figure 5).

Discussion

The activating and inactivating mutations of FSHR areknown to provoke potential alterations in receptor func-tion at different stages. Therefore, the functional studieson the naturally occurring mutations have importance inunderstanding the role of key residues involved in ligand

Figure 2. Stretch of amino acids in human FSHR on either side of Val514 and Ala575. Mutatedresidues in human FSHR are conserved across species and also in human LH/CGR and TSHR.

Figure 3. Western blot showing expression of wild type and mutantFSHR. Lysates obtained from CHO cells stably transfected with emptyvector (mock), wild type or mutant receptors (Val514Ala and Ala575Val)were subjected to SDS-PAGE followed by Western blotting. Upperpanel probed with monoclonal anti-FSHR (106.105) antibody andlower panel probed with anti �-actin antibody (Loading control).



interaction, activation and trafficking of the receptor. Ourgroup had identified two mutations of FSHR gene in twodifferent subjects manifesting distinctly different pheno-types. The present study describes the functional charac-terization of both these mutants with respect to surfaceexpression, binding and signaling.

The novel mutation at position 514 identified in a sub-ject with OHSS developed during IVF was investigated forits functional activity. The OHSS is most often an iatro-genic complication of controlled ovarian hyperstimula-tion treatment. During IVF, in these subjects administra-tion of low amounts of exogenous FSH stimulates thegrowth of multiple follicles, due to the elevated levels ofestrogens leading to OHSS. Although, FSHR gene poly-morphisms have been studied for its association withaOHSS (21–24), the cause of developing aOHSS in IVFsubjects is not yet clear. Moreover, severe form of aOHSScould result in life threatening complication, thus man-agement of hyper responders is one of the challenges in the

field of IVF. So far the activating mutations of FSHR havebeen reported in the subjects who develop sOHSS duringfirst trimester of the pregnancy (25, 26). However, to thebest of our knowledge, there are no reports available in theliterature on the activating mutations of FSHR in subjectswith aOHSS. This is the first report of FSHR mutationleading to Val514Ala substitution in a subjects undergoinggonadotropin stimulation who had developed aOHSS.The basal FSH and LH levels of this subject were observedto be low. It is noteworthy, that on administration of lowamounts of exogenous FSH, the estrogen levels before andafter hCG administration were drastically higher in thissubject. Due to increased levels of estrogen, the number ofpreovulatory follicles and retrieved oocytes were very highas expected, leading to development of aOHSS.

The functional analysis of the mutant receptor 514 wascarried out to understand the impact of mutation on re-ceptor activity. It was observed that the cell surface ex-pression of mutant receptor was very high as compared to

Figure 4. Functional characterization of wild type and mutant FSHR. CHO cells were transfected with expression vector encoding either wild-typeor mutant receptors. A, Surface bound and internalized hormone. B, Internalization index of wild type and mutant FSHR. C, Binding of 125I-FSH tothe cells in the absence or presence of increasing dose of unlabeled FSH. D, FSH-induced cAMP generated. Transfected cells were incubated withincreasing dose of FSH and the cAMP generated was measured. Data represented are mean � SEM of three independent experiments induplicates. ‘*’, ‘**’ and ‘***’ denotes the statistically significant difference at P � .05, P � .005 and P � .0005 respectively.



the WT receptor. Further, it was observed that the bindingof FSH to this mutant receptor was significantly highercompared to WT receptor, which could be due to the in-creased cell surface expression of the mutant receptor. Onthe other hand, the internalization of hormone bound mu-tant receptor was lower as compared to the WT receptorleading to significantly low percent internalization index.No distinct differences were observed in the ligand bindingcharacteristics between the mutant and WT FSHR. Thebasal level of cAMP produced by the cells expressing mu-tant receptor 514 was higher as compared to WT receptor;however this difference was not significant. Therefore, wespeculate that this mutant receptor may not possess any

constitutive activity. However, it was observed that thecAMP levels were significantly higher at lower dose (12.5and 25 ng) of FSH in mutant (514) receptor when com-pared to WT receptor demonstrating hyper-activation ofthe mutant receptor. However, with increasing doses ofFSH the cAMP levels were similar, suggesting that it mighthave reached its saturation at lower dose of FSH (12.5 and25 ng).

It has been reported that in case of sOHSS, the FSHRmutants get activated upon hCG/TSH stimulation, whichsuggest that their ligand specificity could have been af-fected. In contrast, in the present study, the mutant recep-tor 514 showed no response to hCG/TSH stimulation(data not shown). This observation indicates that in caseof aOHSS, FSH itself might trigger the development ofOHSS. Moreover, other factors, which have been reportedto contribute to the pathogenesis of OHSS such as, vas-cular endothelial growth factor (VEGF), cytokines andprostaglandins, remain to be elucidated (27, 28). Also,studies, focusing on the effect of the mutation on the othersignaling pathways needs to be analyzed.

The Valine at position 514 seems to be important forreceptor function as this residue is located in a motif in thecenter of EL2 513KVSIC*PMDV522 which is highly con-served across the FSHRs of different species as well as inLHR and TSHR. Apart from this mutation at position514, mutations at position 512 and 519 have also beenreported in EL2 of FSHR (29, 30). These mutations alterthe trafficking and/or cell signaling ability of the receptor.Therefore, studies with naturally occurring mutations inEL2 might help in delineating its role in various stages ofFSHR function.

The second mutation reported in the present study atposition 575 was identified in a woman with hypergo-nadotropic hypogonadism (15). The resistance to FSH inthis subject could be explained from the characteristics ofmutant receptor 575. The surface localization of the mu-tant receptor 575 was observed to be lower as comparedto WT receptor. Although, in ligand binding studies, thehormone binding characteristics of mutant receptor wassimilar, the amount of ligand bound to the receptor on thecell surface was significantly low. However, the percent-age of receptor internalized was remarkably higher ascompared to WT receptor. Altogether, these observationsindicate defect in trafficking of the mutant receptor to thecell surface. Further, the cAMP levels were almost similarto basal levels even at higher doses of FSH stimulation.This suggests that along with reduced cell surface expres-sion and rapid internalization, the mutation at position575 resulted in reduced signaling activity of the receptor.Recently, it has been demonstrated that the small allostericmolecule (Org41841) can restore the expression of FSHR

Figure 5. Cellular localization of FSHR expression. CHO cells werestably transfected with expression vectors encoding either wild type ormutant Val514Ala and Ala575Val receptors. Cells transfected withvector alone (without FSHR) served as negative control.Nonpermeabilized (left panel) or permeabilized (right panel) cells wereprobed with monoclonal anti-FSHR antibody (106.105) conjugated toAlexa Fluor 568. In the case of Ala575Val mutation, the cell surfaceexpression of FSHR was observed to be very weak. However, uponpermeabilization, the intracellular expression was similar to WT FSHR,indicating that the mutant receptor 575 is defective in cell surfacetrafficking.



mutant Ala189Val on the plasma membrane (31). Similarstudies need to be attempted in future for developing atherapy for subjects affected due to nonfunctionalreceptor.

In this study subject, the ovaries were small with ab-sence of follicles and her uterus was hypoplastic in naturebefore treatment. This subject displayed higher basal FSHand LH levels, which indicated the loss of negative feed-back probably due to inactivation of the FSHR resultingfrom mutation at 575. Therefore, even after giving estro-gen-progestin combined treatment for 9 months, the basalFSH levels of this subject did not reduce to normal. More-over, no follicles were observed in this subject after treat-ment. Nevertheless, she showed development in the breastand uterus after the treatment indicating that her estrogenresponse might be normal. Thus, loss of function of themutant receptor observed by in vitro analysis correlatedwell with the severity of the phenotype observed in thissubject.

The mutation at position 575 is present near the C-tailof the conserved ‘BXXBB’ motif. It has been reported thatthe Alanine substitution of the basic residues in the samemotif present in the C-tail results in reduced surface ex-pression and ligand induced signaling of the mutant re-ceptor (32). Other than the mutation at position 575, mu-tation at position 587 has also been reported to be presentin the TMH-6 region of FSHR. Functional characteriza-tion of this mutation has revealed similar results showingcomplete impairment of the signaling activity of the mu-tant receptor (33). In the interim, these results suggest thatthe residues in the TMH-6 might have crucial role in es-tablishing the structural stability to the receptor, which isessential for its trafficking to the cell membrane and ligandinduced signaling.

The residue at position 575 is conserved across the threeGPHRs and across the mammalian species. It is interestingto note that, the presence of naturally occurring mutationsat the corresponding position in both LHR (at Ala572Val)(34) and TSHR (at Ala627Val) (35) has been previouslydescribed. These substitutions of Ala to Val at both thesepositions resulted in the hyper-activation of the receptor.However, from our in vitro analysis, we conclude that thesubstitution of Ala to Val at position 575 results in inac-tivation of the FSHR. This indicates that the conservedresidues across the three GPHRs might have differentfunctions to play.

In conclusion, the present study provides experimentalevidence towards understanding genotype- phenotypecorrelation with reference to the FSHR mutations de-scribed. In the case of Val514Ala mutant, functional char-acterization showed that the surface expression of the re-ceptor was higher and it gets activated even at low dose of

FSH. This supports the clinical manifestation observed inan IVF subject presenting Val514Ala mutation, whose es-trogen levels before and after hCG administration wereincreased on administration with low dose of exogenousFSH. This has probably resulted in increased number offollicles and retrieved oocytes further developing OHSS.On the other hand, in the case of Ala575Val mutant, weobserved that the receptor was defective in cell surfacetrafficking. Due to this, its cell signaling activity was alsoobserved to be impaired leading to inactivation of the re-ceptor. These findings suggest that the mutation Ala575Valcould be the probable cause for the primary amenorrheaobserved in the studied subject. Thus, our results suggestthat the residues Val514 and Ala575 seem to be importantfor exhibiting optimum receptor activity.

Acknowledgments

We thank Dr. Anurupa Maitra, Dr. Srabani Mukherjee and Ms.Nanda Ugale (DNA sequencing core facility, NIRRH) for theirassistance in DNA sequencing. We thank Dr. Nafisa Balasinor,Ms. Shobha Sonawane and Ms. Reshma Goankar in facilitatingconfocal microscopy analysis. Authors acknowledge the kindgift of 106.105 monoclonal anti-FSHR antibody provided by Dr.J. A. Dias, Wadsworth Center, David Axelrod Institute for PublicHealth, Albany, NY. We sincerely thank Dr. M G R Rajan andMs. Bharati Jain, Radiation Medicine Centre (RMC) of BhabhaAtomic Research Centre (BARC), Mumbai for the kind gift ofrecombinant hTSH. We also thank Mr. Rahul Mojidra for thetechnical assistance provided. Authors acknowledge the valu-able assistance provided by Dr. Ripal Gevariya, Fertility Clinicand IVF Centre to obtain additional clinical information.

Address all correspondence and requests for reprints to: SmitaD. Mahale, Division of Structural Biology and ICMR BiomedicalInformatics Centre, National Institute for Research in Repro-ductive Health, Indian Council of Medical Research, Mumbai400 012, India, Tel: �91–22–21 492 015, Fax: �91–22–24 139 412, E-mail: [email protected].

3Correspondence: Tel: �91–22–21 492 015; Fax: �91–22–24 139 412; e-mail: [email protected]

Reprint request: Smita D. Mahale, Division of Structural Bi-ology and ICMR Biomedical Informatics Centre ., National In-stitute for Research in Reproductive Health, Indian Council ofMedical Research, Mumbai- 400 012, India, Tel: �91–22–21 492 015, Fax: �91–22–24 139 412, E-mail:[email protected]

Disclosure summary: The authors have nothing to disclose.Precise: The functional characterization of FSHR mutations

at position 514 and 575 revealed that, these mutations eitheralter expression or signaling activity of the receptor.

This work was supported by This study (NIRRH/RA/190/09–2014) was supported by grants from Indian Council of Med-ical Research, New Delhi, India (P & I/BIC/1/1/2009) and Boardof Research in Nuclear Sciences and Department of Atomic En-ergy. Ms. Swapna Desai was supported by the Senior Research



mailto:[email protected]



Fellowship (IRIS No: 2011–00 850) from Indian Council ofMedical Research, New Delhi, India.

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