The n e w e ng l a n d j o u r na l of m e dic i n e

original article

A Genetic Basis for Functional
Hypothalamic Amenorrhea
Lisa M. Caronia, B.A., Cecilia Martin, Ph.D., Corrine K. Welt, M.D.,
Gerasimos P. Sykiotis, M.D., Ph.D., Richard Quinton, M.D.,
Apisadaporn Thambundit, B.A., Magdalena Avbelj, M.D., Ph.D.,
Sadhana Dhruvakumar, M.Sc., Lacey Plummer, B.A., Virginia A. Hughes, M.Sc.,
Stephanie B. Seminara, M.D., Paul A. Boepple, M.D., Yisrael Sidis, Ph.D.,
William F. Crowley, Jr., M.D., Kathryn A. Martin, M.D., Janet E. Hall, M.D.,
and Nelly Pitteloud, M.D.

A BS T R AC T

Background
Functional hypothalamic amenorrhea is a reversible form of gonadotropin-releasing From the Harvard Center for Reproduc-
tive Endocrine Sciences and Reproductive
hormone (GnRH) deficiency commonly triggered by stressors such as excessive exer- Endocrine Unit and the Department of
cise, nutritional deficits, or psychological distress. Women vary in their susceptibility Medicine, Massachusetts General Hospi-
to inhibition of the reproductive axis by such stressors, but it is unknown whether tal — both in Boston (L.M.C., C.M., C.K.W.,
G.P.S., A.T., M.A., S.D., L.P., V.A.H., S.B.S.,
this variability reflects a genetic predisposition to hypothalamic amenorrhea. We P.A.B., Y.S., W.F.C., K.A.M., J.E.H., N.P.);
hypothesized that mutations in genes involved in idiopathic hypogonadotropic hypo- the Department of Endocrinology, Royal
gonadism, a congenital form of GnRH deficiency, are associated with hypothalamic Victoria Infirmary, and the Institute for
Human Genetics, University of Newcastle
amenorrhea. upon Tyne — both in Newcastle upon
Tyne, United Kingdom (R.Q.); and the En-
Methods docrine Division, Centre Hospitalier Uni-
We analyzed the coding sequence of genes associated with idiopathic hypogonado- versitaire Vaudois, University of Lausanne,
Lausanne, Switzerland (Y.S., N.P.). Address
tropic hypogonadism in 55 women with hypothalamic amenorrhea and performed in reprint requests to Dr. Pitteloud at the
vitro studies of the identified mutations. Endocrine Division, Centre Hospitalier
Universitaire Vaudois, University of Laus-
Results anne, Rue du Bugnon 46, CH-1011 Laus-
anne, Switzerland, or at nelly.pitteloud@
Six heterozygous mutations were identified in 7 of the 55 patients with hypotha- chuv.ch.
lamic amenorrhea: two variants in the fibroblast growth factor receptor 1 gene
FGFR1 (G260E and R756H), two in the prokineticin receptor 2 gene PROKR2 (R85H Ms. Caronia and Dr. Martin contributed
equally to this article.
and L173R), one in the GnRH receptor gene GNRHR (R262Q), and one in the Kall-
mann syndrome 1 sequence gene KAL1 (V371I). No mutations were found in a cohort N Engl J Med 2011;364:215-25.
of 422 controls with normal menstrual cycles. In vitro studies showed that FGFR1 Copyright © 2011 Massachusetts Medical Society.

G260E, FGFR1 R756H, and PROKR2 R85H are loss-of-function mutations, as has been
previously shown for PROKR2 L173R and GNRHR R262Q.

Conclusions
Rare variants in genes associated with idiopathic hypogonadotropic hypogonadism
are found in women with hypothalamic amenorrhea, suggesting that these muta-
tions may contribute to the variable susceptibility of women to the functional
changes in GnRH secretion that characterize hypothalamic amenorrhea. Our ob-
servations provide evidence for the role of rare variants in common multifactorial
disease. (Funded by the Eunice Kennedy Shriver National Institute of Child Health
and Human Development and others; ClinicalTrials.gov number, NCT00494169.)

n engl j med 364;3  nejm.org  january 20, 2011 215
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hypothalamic amenorrhea were present. All rights reserved. The mean age at men- hypogonadism is characterized by an absence of arche was 13. with 13 patients report- puberty and by infertility. low se- sumes in most cases. and 9 a fam- variable expressivity of the clinical features of ily history of hypothalamic amenorrhea.15 The involved genes disorder characterized by dietary restriction and encode proteins essential for GnRH neuron devel. caused by defects in ing delayed menarche (age at onset.20 None of the patients met the diagnostic lamic amenorrhea.1 excessive exercise. All patients had normal results terizes hypothalamic amenorrhea. All 55 patients with hypothalamic amenorrhea tal GnRH deficiency (idiopathic hypogonado. 2011 The New England Journal of Medicine Downloaded from nejm. had a body-mass index (BMI. site sensitivity of the GnRH pulse generator to en- ergy deficits is evidenced by the fact that serum Patients with Hypothalamic Amenorrhea levels of leptin.4±6.8-10 After the underlying stressors have history of secondary amenorrhea for 6 months or been eliminated. and 28 had a subclinical eating proximately 40% of cases. defined as to 32 days’ duration) for the previous 2 years. and the lamic amenorrhea. which is a pre- hypothalamic–pituitary–gonadal axis.2 eating disorders. and 39±25 pg per milliliter (143±92 pmol per tional deficiency in GnRH secretion that charac.5±1.3  nejm. No other uses without permission.org  january 20. (±SD) age at diagnosis was 22. and defense mechanisms have evolved that temporar. and administration of exogenous 375 of the 422 women.org on June 7.15 The disease is genetically heterogeneous. 20 with several associated loci that account for ap. preoccupation with weight. These factors included excessive exer- hibition of the hypothalamic–pituitary–gonadal cise (>5 hours per week).3 and psychological distress4 sup. and the absence of menses. it is unknown whether this susceptibil. and hypoestrogenemia without organic grams divided by the square of the height in me- abnormality.7 The exqui. had had a normal menstrual cycle (27 functional hypothalamic amenorrhea. criteria for anorexia nervosa at presentation.5 This undergone normal puberty (menarche at ≥10 but frequent cause of female infertility is diagnosed as <15 years). as ascertained with the use of the Eating Attitudes ity reflects a genetic predisposition to hypotha. had weight loss.12.21 Six patients had opment and GnRH secretion and action. butions of multiple genetic defects or epigenetic follicle-stimulating hormone (FSH). The mean tropic hypogonadism).11 Among both women and rum estradiol levels. had completed puberty spontaneously. a signal of fat reserves. recruited by means of advertising. The n e w e ng l a n d j o u r na l of m e dic i n e R eproduction is an energetically Me thods costly process for women.8 years. Stressors such as weight loss. the weight in kilo- pin levels. are often Hypothalamic amenorrhea was diagnosed in 55 low in patients with hypothalamic amenorrhea women presenting to the Massachusetts General and that leptin replacement can restore GnRH Hospital or Newcastle upon Tyne Hospital with a pulsatility. ing factors.20. ters) between 18 and 35. for hypothalamic amenorrhea were reported for tion patterns. 2017. respectively.19 loss of more than 15% axis by such stressors varies substantially. Controls press the hypothalamic–pituitary–gonadal axis Controls were 422 women from the greater Boston by inhibiting hypothalamic pulsatile secretion of area.3 IU per li- hypogonadism confer susceptibility to the func. more. We hypothesized that mutations diol in the group with hypothalamic amenor- in genes involved in idiopathic hypogonadotropic rhea were 4. For personal use only.16-18 The a family history of delayed puberty. low or normal gonadotro. Copyright © 2011 Massachusetts Medical Society. sensitivity to the in. in contrast to hypotha. ≥15 years) at the secretion of GnRH from the hypothalamus a time when no factors predisposing them to or defects in the action of GnRH on the pitu. liter).2. on neuroimaging.1 years. No predisposing factors ciated with a spectrum of abnormal GnRH-secre. and none had symptoms or 216 n engl j med 364. 6. the remaining 47 exercised pulsatile GnRH can restore functionality of the for more than 5 hours per week. .6 Hypothalamic amenorrhea is asso. Idiopathic hypogonadotropic mean BMI was 19.0 IU per liter.7±3. mean serum levels of luteinizing hormone (LH). low or normal gonadotropin levels. disposing factor.4±2.1±3.14. Study Participants ily inhibit reproduction under adverse conditions. and estra- perturbations. who had gonadotropin-releasing hormone (GnRH). and one or more predispos- female nonhuman primates. and a subclinical eating disorder However. The GnRH deficiency most likely reflects the contri. Test.21 Much is known about the genetics of congeni. All participants provided written informed consent. five patients reported exercising excessively. Twenty- itary.13 of body weight. normal reproductive function re. ter.

27 the (FGFR1. No other uses without permission. as previously described. No as reported by study physicians.29. hyperandrogenemia. The FGFR1 G260E and R756H mutants ity to activate downstream signaling was com. All rights reserved. All 7 patients were white.30 The osteocal. fied as previously described. For personal use only. acne.23 the GnRH receptor gene GNRHR. The abil. in separate polymerase-chain-reaction assays.3  nejm. Four-parameter sigmoidal dose–­ tients underwent LH-secretion studies involving response curves were generated and analyzed blood sampling every 10 minutes over a 24-hour with the use of Prism 4 statistical software period. and KAL1) were identi- prokineticin 2 gene PROK2. and normal results on neuroimaging. whole-cell extracts.24 the G protein–coupled receptor 54 gene GPR54. in triplicate. tant and wild-type receptors were compared by otropic hypogonadism were determined in all means of Student’s two-tailed t-test. and R756H mutants result in loss of function.29. 95% confidence interval. respectively. These genetic rare sequence variants were found in a cohort of variants were absent among the controls (the 375 422 controls with normal menstrual cycles. GNRHR. Each assay was performed twice an LH-to-FSH ratio >1). as previously described.26 idiopathic hypogonadotropic hypogonadism the fibroblast growth factor 8 gene FGF8. ognition by the antibody (Clone HA-7. or ing.29 and gand domain and tyrosine kinase domain of the were studied in L6 myoblasts and human embry. Sigma). mitogen-activated protein kinase (MAPK) signal- drome (hirsutism. of a transcriptional assay show that the G260E cin fibroblast growth factor (FGF) response ele. respectively. Total expression of wild-type and mutant FGFR1 nadotropic hypogonadism. and PROKR2 transfected into COS-7 cells was de- complete puberty as of 18 years of age.25 the Heterozygous mutations in genes associated with fibroblast growth factor receptor 1 gene FGFR1. samples from patients with hypothalamic amen- orrhea: the Kallmann syndrome 1 sequence gene R e sult s KAL1.001) (Fig.11.28 and the prokineticin fied in 7 of the 55 patients with hypothalamic receptor 2 gene PROKR2. 2L and 2M).29 Sequence variations amenorrhea (13%.org  january 20.32 findings that are consistent with dis- PROKR2 R85H mutation were introduced into ease-associated mutations. respectively. Genetic Basis for Hypothalamic Amenorrhea biochemical signs of the polycystic ovary syn. All had absent or in. Genetic Studies with the use of a 3xHA tag sequence fused with Genomic DNA was extracted from peripheral. otherwise normal anterior pituitary The cell-surface expression of FGFR1 was quanti- function. 2I and 2J). previously described expression vectors by means FGFR1 mutants (G260E and R756H) in the li- of a QuickChange XLII Kit (Stratagene)28. low serum es. . results assays. as ment (OCFRE) reporter and the murine early demonstrated by a decrease in FGF-induced MAPK growth response 1 (Egr-1) reporter served as indi. 2F and 2G) and on wild-type counterpart in transient transfection the cell surface (Fig. the N-terminal end of PROKR2 to facilitate rec- blood samples obtained from all participants. A subgroup of the pa. 5 to were found on both strands and were confirmed 24) (Fig. Copyright © 2011 Massachusetts Medical Society.org on June 7. PROKR2. Gene-Reporter Assays 2D. 1 and Table 1). 2011 217 The New England Journal of Medicine Downloaded from nejm. n engl j med 364. receptor. However. both overall (Fig. Ex. low or termined by means of Western blotting involving normal serum gonadotropin levels.29.22 (GraphPad).30 tradiol levels. 2C. The variants alter amino acids and PROKR2 Mutations that are highly conserved across species (Fig. reporter activity (reflected by OCFRE activity) cators of FGFR1-induced and PROKR2-induced (P<0.30 and the cell-sur- face expression of PROKR2 was also quantified. showed expression levels similar to those of wild- pared between the mutated receptor and its type FGFR1. onic and proximal intronic sequences (located at Antibody-binding assays were performed three least 15 bp from the splice sites) of seven genes times in quadruplicate. Patients with Idiopathic Hypogonadotropic Total Expression and Cell-Surface–Receptor Hypogonadism Expression We evaluated 160 women with idiopathic hypogo. women without risk factors for hypothalamic amenorrhea and the 47 women who exercised >5 Functional Characterization of New FGFR1 hours per week). and 2. were found in Patients 1 onic kidney (HEK) 293 cells. Expression levels of mu- implicated in the cause of idiopathic hypogonad.31. and 2E) and cause considerable loss of func- The FGFR1 G260E and R756H mutations and the tion. 2017.

circles female family members.2). the seven patients is given in the Supplementary All seven patients with hypothalamic amenor. 2011 The New England Journal of Medicine Downloaded from nejm. and diamonds offspring whose sex is not shown (with the numbers of persons given within the diamond). For personal use only. R756H. Squares indicate male family members. The n e w e ng l a n d j o u r na l of m e dic i n e Patient 1 Patient 2 Patient 3 2 FGFR1 G260E/+ FGFR1 R756H/+ PROKR2 R85H/+ +/+ 4 PROKR2 R85H/+ 3 Patient 4 Patient 5 Patient 6 GNRHR +/+ PROKR2 +/+ L173R/+ +/+ GNRHR R262Q/+ GNRHR R262Q/+ +/+ PROKR2 L173R/+ +/+ L173R/+ Patient 7 HA Delayed puberty HA and delayed puberty Adopted Lost pregnancy KAL1 V371I/+ Mutation carrier Figure 1.org). L173R. rhea for at least 6 months and at least one risk Our patients with hypothalamic amenorrhea factor for hypothalamic amenorrhea (Table 2). R262Q. The PROKR2 mutant R85H identified in Patient Four of the seven reported a family history of hy- 3 is also a loss-of-function mutant. and the BMI at diagnosis ranged from pression and signaling activity) (Fig. All rights reserved.4±1.4±6. No other uses without permission. 2017. Four of the seven pa- and 2N). ticle at NEJM. as evidenced by pothalamic amenorrhea or delayed puberty (Fig. Copyright © 2011 Massachusetts Medical Society. the age decreased signaling activity as compared with at diagnosis ranged from 18 to 34 years (mean. A more detailed summary of for KAL1. who had the PROKR2 R85H or FGFR1 R756H mu- 218 n engl j med 364. 1 decreased overall and cell-surface expression and and Table 2).org on June 7. with plus signs indicating wild-type and G260E. The other tient 7 could not be assessed (Table 2) owing to five discontinued hormonal therapy and had the scarcity of in vitro functional assays available recovery of menses.31 The PROKR2 L173R mutant seen in tients had attempted to conceive.org  january 20.9). . Pedigrees of the Seven Patients with Hypothalamic Amenorrhea (HA) Found to Have Mutations. Among the seven patients. the patient with the mutation is indicated by a red arrow. tempts were successful. 2H. wild-type PROKR2 (P<0. 24. and V371I indicating the amino acid mutations.3  nejm. R85H. with one patient conceiv- tients 5 and 6 have previously been reported as ing without assisted reproductive treatment. three of the at- Patient 4 and the GNRHR R262Q mutant in Pa.29. 2K. and the allele status is given below the proband or affected family members.001 for cell-surface ex. 18 to 22 (mean. 19. Appendix (available with the full text of this ar- rhea who had mutations had secondary amenor. of the seven patients continued to receive long- characteristics of the KAL1 V371I mutant in Pa.32 Finally. The mutated gene is indicated in bold to the left of the ped- igree. For each pedigree. term hormone-replacement therapy. Two loss-of-function mutants (Table 2).31.

Genetic Basis for Hypothalamic Amenorrhea Table 1.7 We speculate that defects. However.pathic hypogonadotropic hypogonadism was tra- gonadotropic hypogonadism. This finding expands our understanding of R85H and L173R mutations described here have the genetics of GnRH-deficiency disorders. rons. No other uses without permission. FGFR1 and PROKR2 sig- is activated by gonadotropin-releasing hormone 1 naling also modifies eating behavior in mice. and survival of GnRH-secreting neu.31 The GNRHR R262Q mutant was studied by de Roux and colleagues. 3). Idio- previously been associated with idiopathic hypo. For personal use only.38.idiopathic hypogonadotropic hypogonadism also pathic hypogonadotropic hypogonadism).37 ditionally considered a genetically determined. a suboptimal maturation of the GnRH network during puberty. 2017. According to Group.org on June 7. and FGFR1 controls the fate specification. All rights reserved. Patients who had hypothalamic amenorrhea as congenital.32. mutations in these genes un. even if they have genetic hypothalamic amenorrhea. The functional activity of PROKR2 L173R was evaluated by Cole and colleagues29 and Monnier and colleagues. and lifelong form of GnRH deficien- well as the PROKR2 R85H or FGFR1 R756H muta. Frequency of Loss-of-Function Mutations in the Study Participants.36. thalamus. as previously described in women with ment is discontinued. or a defective regulation Discussion of GnRH secretion — since both proteins are expressed not only during development but also We found genetic defects in several patients with in the adult hypothalamus.thalamic amenorrhea.35. Patients with Controls Controls Patients with Idiopathic Who Were Who Were Hypothalamic Hypogonadotropic Menstruating but Menstruating and In Vitro Amenorrhea Hypogonadism Not Exercising Exercising >5 Hr/ Mutation Functionality* (N = 55) (N = 160) (N = 375) Wk (N = 47) no.org  january 20. We found that genes mutated in patients with derlie severe congenital GnRH deficiency (idio. as many as 10% of patients with tion in our study were also shown to have abnor. Copyright © 2011 Massachusetts Medical Society. in hypothalamic amenorrhea.the GnRH network and its sensitivity to nonge- work owing to a smaller-than-normal number netic factors. .cy. KAL1 and PROKR2 are Thus. we speculate that genetic defects in these critical for the migration of GnRH-secreting neu.39 (GnRH1) in the pituitary33.28 This would. The affected genes turn.idiopathic hypogonadotropic hypogonadism re- mal patterns of endogenous GnRH-induced LH sume normal reproductive function after treat- secretion.15 In are mutated in those with hypothalamic amenor- fact. of participants FGFR1 R756H Decreased 1 0 0 0 G260E Decreased 1 0 0 0 PROKR2 R85H Decreased 1 0 0 0 L173R Decreased 1 5 0 0 GNRHR R262Q Decreased 2 3 0 0 KAL1 V371I Not assessed 1 0 0 0 * The functional activity of the FGFR1 R756H and G260E mutants and the PROKR2 R85H mutant was assessed in this study. 2011 219 The New England Journal of Medicine Downloaded from nejm.3  nejm. predispose persons to abnormal GnRH se- play fundamental roles in GnRH ontogeny and cretion under the influence of factors that stress function: GNRHR encodes the unique receptor that the reproductive system. Conversely.16.32 tation also had abnormal patterns of endogenous completed embryonic migration to the hypo- GnRH-induced LH secretion (Fig.31.17 In humans.40 This reversal of idiopathic hypogonado- decreased PROKR2 or FGFR1 signaling leads to tropic hypogonadism indicates the plasticity of a partially compromised GnRH neuronal net.pathways may also contribute to the abnormal rons34. the GNRHR R262Q mutation and the PROKR2 rhea. idiopathic hypogonad- of GnRH-producing cells that have successfully otropic hypogonadism occasionally is present in n engl j med 364. eating patterns seen in many patients with hypo- migration.29.

All rights reserved.org on June 7.3  nejm. 2017. 220 A B PROKR2 FGFR1 1 NH2 1 D1 D2 D3 TM Tyrosine kinase domain 822 76 88 384 G260E R756H R85H COOH C G260 D R756 E R85 Human ILQA G LPAN Human VEDLD R IVALT Human RYKKL R NLTNL Mouse ILQA G LPAN Mouse VEDLD R IVALT Mouse RYKKL R NLTNL Dog ILQA G LPAN Dog VEDLD R IVALT Dog RYKKL R NLTNL Puffer fish ILQA G LPAN Puffer fish VEDLD R CLAMT Puffer fish RYKKL R NLTNL Xenopus ILQA G LPAN Xenopus VEDLD R LVALS Xenopus RYKKL R NLTNL e 0E e 6 H e ild yp 26 ild yp 75 ild yp 5H F EV W T G G EV W T R H EV W T R8 The FGFR1 FGFR1 PROKR2 HSP90 HSP90 HSP90 I J K P<0.org  january 20. 100 100 100 m e dic i n e FGFR1 80 80 80 G260E PROKR2 60 60 FGFR1 60 R85H R756H 40 40 40 Downloaded from nejm. 2011 Wild-type FGFR1 Wild-type FGFR1 Wild-type PROKR2 Copyright © 2011 Massachusetts Medical Society. (% of wild type) (% of wild type) (% of wild type) Egr-1–Luc Activity OCFRE-Luc Activity OCFRE-Luc Activity 20 20 20 EV EV EV 0 0 0 0 10−13 10−12 10−11 10−10 10−9 10−8 0 10−13 10−12 10−11 10−10 10−9 10−8 0 10−9 10−8 10−7 10−6 10−5 FGF8 Level (M) FGF2 Level (M) PROK2 Level (M) .001 120 120 120 100 100 100 80 80 80 60 60 60 40 40 40 (% of wild type) (% of wild type) (% of wild type) 20 n e w e ng l a n d j o u r na l 20 20 The New England Journal of Medicine of Cell-Surface Antibody Binding Cell-Surface Antibody Binding Cell-Surface Antibody Binding 0 0 0 EV Wild-Type FGFR1 EV Wild-Type FGFR1 EV Wild-Type PROKR2 FGFR1 G260E FGFR1 R756H PROKR2 R85H L M N 120 120 120 n engl j med 364. No other uses without permission. For personal use only.

T and I bars indicate standard errors nadotropic hypogonadism. Genetic Basis for Hypothalamic Amenorrhea ment therapy.36. the ligand-binding domains of FGFR1. the genetic component of hypo- Figure 2 (facing page). If hypothalamic amenorrhea has a genetic terest. helping to balance survival against in genes underlying idiopathic hypogonadotropic the metabolic needs of pregnancy. hormonal. NELF [the the wild type (P<0. energy availability. and H show that the overall expression levels of FGFR1 G260E and R756H were normal. including amenorrhea. but is Panels A and B show the structures of FGFR1 and not sufficient to cause. and E show that the FGFR1 G260 basis partially in common with idiopathic hypo- and R756 amino acids and the PROKR2 R85 amino gonadotropic hypogonadism. D1.37 shown that stress can induce reproductive dys. Loss-of-Function Mutations in Patients with Hypothalamic Amenorrhea. 2017.42 Furthermore.01).19 Our findings may help to explain the genes associated with idiopathic hypogo- the variable susceptibility of women to inhibi. clinical phenotypes? The total load of mutations whereas PROKR2 R85H expression levels were de. or PROKR2.36. . This explana- hypogonadism27 have normal reproductive func.org  january 20. and in many of these cases was inherited from Studies of humans and nonhuman primates have an asymptomatic parent. and N show genic inheritance of mutations associated with that the FGFR1 G260E mutant has decreased fibro- idiopathic hypogonadotropic hypogonadism blast growth factor 8 (FGF8)–induced osteocalcin FGF response element (OCFRE) activity as compared with (FGFR1 in combination with GNRHR.12. mostly in the heterozygous state.9.25. for gel loading. PROKR2. and that the pairs) has been reported. Twen- Since patients with mutations resumed regular ty-five percent of women with hypothalamic menses after discontinuing hormone-replace. suggesting that their GnRH mutations associated with idiopathic hypogo- deficiency results from a combination of genetic nadotropic hypogonadism and hypothalamic and environmental influences. 2011 221 The New England Journal of Medicine Downloaded from nejm.001). while not sufficient to cause idiopathic hypogo- main of FGFR1. ase reporter vector. could set a lower of the means of two experiments performed in tripli- threshold for functional inhibition of the hypo- cate for the gene-reporter assays or the means of three experiments performed in quadruplicate for the anti. in genes related to GnRH ontogeny and action creased (P<0. adult men (in which case it is called adult-onset Such a lower threshold for inhibition might also idiopathic hypogonadotropic hypogonadism). it will be impor- though not all.001). Luc the lucifer. amenorrhea in our study had a history of delayed n engl j med 364. An empty vector (EV) was used as a Patients with idiopathic hypogonadotropic hypo- negative control for protein expression. Panels L. D. and D3 denote lamic amenorrhea in our study were heterozygous.13.org on June 7. G. Copyright © 2011 Massachusetts Medical Society. Panels C. Panels I. M. nutritional.43 PROKR2 R85H mutant has decreased PROK2-induced All six mutations associated with hypotha- early growth response 1 (Egr-1) activity as compared with the wild type (P<0. No other uses without permission. hormone factor]. We speculate that such heterozygous mutations. thalamic–pituitary–gonadal axis under adverse body-binding assays. or both. For personal use only.37.32. in many.36.001). respectively. females with normal menstrual tant to undertake comprehensive sequencing of cycles.001). nadotropic hypogonadism in larger cohorts of tion of the hypothalamic–pituitary–gonadal axis.30. All rights reserved. amenorrhea in persons who do not have symp- In contrast to idiopathic hypogonadotropic toms. The PROKR2 L173R mutation appears to be hypogonadism. and K show that the receptor cell-surface expression levels gonadism frequently show homozygosity and in COS-7 cells were similar to the wild-type levels for compound heterozygosity for mutations at the both FGFR1 mutants but were significantly decreased disease-causing loci. To elucidate the genetics of hypothalamic function. psychological stress. GnRH deficiency. as well as the mutations of in. what are the fac- acid are highly conserved across vertebrate species.31.3  nejm. might be less in hypothalamic amenorrhea than Heat-shock protein 90 (HSP90) was a positive control in idiopathic hypogonadotropic hypogonadism. patients with hypothalamic amenorrhea. or other digenic pared with the wild type (P<0.41 confer a selective advantage to female carriers dur- Some men with the disease who carry mutations ing famine. tors that ultimately generate these divergent Panels F. J. amenorrhea more completely. hypothalamic amenorrhea has an example: it has been reported in more than a been traditionally viewed as a functional form of dozen patients with idiopathic hypogonadotropic GnRH deficiency resulting from insufficient hypogonadism. that the FGFR1 R756H mutant gene encoding the nasal embryonic LH-releasing has decreased FGF2-induced OCFRE activity as com. tion would be consistent with the presence of tion before onset. or psychological conditions and thereby lead to hypothalamic amenorrhea. as compared with wild-type levels. D2. thalamic amenorrhea predisposes one to. di- for PROKR2 R85H (P<0. al. and TM the transmembrane do.

overall protein expression. Results of Clinical and Genetic Studies in the Seven Patients with Hypothalamic Amenorrhea with Rare Variants in Genes Associated with Idiopathic Hypogonadotropic Hypogonadism. No other uses without permission. § For Patient 4. Cell-surface expression‡ Similar to wild Similar to wild Decreased Decreased§ NA NA NA m e dic i n e type type Signaling activity‡ Decreased Decreased Decreased Decreased§ Decreased¶ Decreased¶ NA * GnRH denotes gonadotropin-releasing hormone. and NA not assessed. No attempt at No attempt at Conceived while conception while receiv. 2017.org  january 20. cell-surface-receptor expression.5 17 19 18 22 22 19 The Predisposing factors Weight loss Yes Yes Yes Yes Yes Yes Yes Subclinical eating disorder No Yes Yes Yes No No Yes Excessive exercise No Yes No No No No Yes Fertility status No attempt at Failed to conceive Conceived while Conceived with. out therapy conception conception receiving go- ing GnRH satile GnRH nadotropin therapy therapy therapy Recovery of menses NA Yes Yes Yes Yes NA Yes Family history of hypothalamic amenorrhea No No Yes No No Yes Yes n e w e ng l a n d j o u r na l The New England Journal of Medicine Genetic and functional characteristics of Gene and variant identified FGFR1 G260E FGFR1 R756H PROKR2 R85H PROKR2 L173R GNRHR R262Q GNRHR R262Q KAL1 V371I Overall protein expression‡ Similar to wild Similar to wild Decreased Decreased§ NA NA NA n engl j med 364. 2011 type type Copyright © 2011 Massachusetts Medical Society. transcriptional level is based on the study by de Roux and colleagues.5 15 12 14 16. 222 Table 2.* Characteristic Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Clinical characteristics Age (yr) At menarche 16 13. Downloaded from nejm.31 ¶ For Patients 5 and 6. receiving pul.32 . and transcriptional level are based on the studies by Cole and colleagues29 and Monnier and colleagues. All rights reserved.org on June 7. For personal use only. and protein expression and signaling activity in Patient 7 were not assessed owing to the scarcity of in vitro functional assays for KAL1.5 15 At diagnosis of hypothalamic amenorrhea 19 28 26 18 34 18 28 BMI at diagnosis† 18. † The body-mass index (BMI) is the weight in kilograms divided by the square of the height in meters. ‡ Protein expression in Patients 5 and 6 was not assessed because of the loss-of-function nature of the mutations.3  nejm.

20 estradiol. 2011 223 The New England Journal of Medicine Downloaded from nejm. 4.45 Unlike our Genetic defects within pathways controlling study. . All rights reserved.3 IU/liter. as apulsatile in a patient with hypothalamic amenorrhea and the FGFR1 R756H mutation (Panel B). The members of patients with idiopathic hypogo. The pattern of LH secretion induced by endogenous gonadotropin-releasing hormone in the early follicular phase of the menstrual cycle is shown as a normal.org on June 7. 2017. multiply by 3. and as both apulsatile and pulsatile with increasing amplitude during the night in a patient with the PROKR2 R85H mutation (Panel C). 20 estradiol. neuro- gonadism or hypothalamic amenorrhea also trophic tyrosine kinase receptor types 2 and 3. leptin.org january 20. Mean levels of LH. No other uses without permission.671. genetic susceptibility to anorexia or bulimia nadotropic hypogonadism. and estradiol are listed for the two patients. For personal use only. 54 pg/ml LH (IU/liter) 15 Apulsatile 10 5 0 8:00 12:00 16:00 20:00 0:00 4:00 8:00 Clock Time (hr) C PROKR2 R85H 25 LH. pulsatile pattern in a healthy woman (Panel A).4 IU/liter. which is frequently seen in family contribute to hypothalamic amenorrhea. which sought rare genetic variants. puberty. and hypocretin. Copyright © 2011 Massachusetts Medical Society. serotonin. Patterns of Luteinizing Hormone (LH) Secretion over a 24-Hour Period. Genetic Basis for Hypothalamic Amenorrhea A Wild Type 25 Nighttime LH (IU/liter) 20 15 10 5 0 8:00 12:00 16:00 20:00 0:00 4:00 8:00 Clock Time (hr) B FGFR1 R756H 25 LH. as brain-derived neurotrophic factor. contribute to delayed puberty. follicle-stimulating hormone (FSH).0 IU/liter. Arrowheads indicate peaks in secretion (of which there are none in the apulsatile pattern in Panel B). FSH. FSH. the appetite or stress-response systems might also prior studies focused on common DNA polymor- n engl j med 364. To convert values for estradiol to picomoles per liter. 9. 1.2 IU/liter. According to Mutation Status. It may be worthwhile nervosa has been investigated in several associa- to investigate whether rare variants in genes tion studies that examined candidate genes such underlying idiopathic hypogonadotropic hypo. 4.44. 47 pg/ml LH (IU/liter) 15 Nighttime 10 Pulses 5 0 8:00 12:00 16:00 20:00 0:00 4:00 8:00 Clock Time (hr) Figure 3.3 nejm.

et al. The GPR54 gene as a regulator of weight women with hypothalamic amen. previous menstrual function and exercise and eating pathology in college 2. Endocr Rev 1998. we would not recommend Clinical and Translational Science Center). and isolated anosmia occurring 224 n engl j med 364. netic considerations. Crowley WF Jr. the full text of this article at NEJM.8:334- function in girls.12:871-8.185: 11. pathic hypogonadotropic hypogonadism. the Newcastle Uni- that women with hypothalamic amenorrhea be versity Teaching Hospitals Special Trustees. Hall JE. Acierno JS Jr.7:11-23. The importance of autosomal col 1986. N Engl J Med pathway in reproduction. 6.86:1580-8.155:531-43. phenotypic spec- 8. Copyright © 2011 Massachusetts Medical Society. J Neuroendocri- mum weight for height necessary for their 2004. Crowley WF spite desensitization at fast pulse fre- J Clin Endocrinol Metab 1989. Wilson GT. et 12. 2001. Martin KA. J Clin Endocrinol women: diagnosis and therapy with pul. Science 1974. et al. Warren MP. Bethea CL. ous exercise in untrained women. Dwyer AA. Chatzidaki E.349:1614-27. 4. Sanchez RL. trum. most likely owing to small sample sizes. Eat Behav 2007. Hayes FJ. Thome JL. Am J Obstet Gyne. Centeno ML. N Engl J Med 1974. Prevalence. Alterations in the hypothalamic-­ stress. maintenance or onset. 13. 49.20:141-63.68:402-11. Novak M.83:2309-12. No other uses without permission. ciency in the human (idiopathic hypogo. McNicholl DJ. Filicori M. puberty. 24. Diversity in fibroblast growth factor re. Finkelstein JW. Crowley WF Jr. 84:1028-36. 19. environment. superior to luteinizing hormone as a mark- pituitary-ovarian and the hypothalamic. Reindollar RH. Bédécarrats Endocr Rev 1986. Bullen J. Agras WS.351:987-97. von Garfinkel PE. Metab 2001. J Clin Endocrinol Metab 1999. Seminara SB. References 1. McArthur JW. Kiss.312:1349-53. 16. J Neuroendocrinol 2007. phenotype correlations and neuroendo- Hypogonadotropic disorders in men and 15. 604. Psychol Med 1982. Yen monkeys with different sensitivity to Marsh EE. Seminara SB. Endocri. Cameron JL. blast growth factor 8 signaling through otropin-releasing hormone receptor mu- old of energy availability in regularly fibroblast growth factor receptor 1 is re. Meysing and nutritional intake. Kim SH. we demonstrated that patients tional Institutes of Health (NIH) (through cooperative agree- ment 5U54HD028138 as part of the Specialized Cooperative with hypothalamic amenorrhea have mutations Centers Program in Reproduction and Infertility Research and in genes regulating GnRH ontogeny and action. Obligatory 949-51. Re. Reversible Kallmann syndrome. Perkins RB. Fibro. orrhea: the effects of body composition ceptor 1 regulation: learning from the 26.5:569-76. Garner DM. Luteinizing hor. GY. McDonough PG. Beranova a study of 262 patients. 9. nol 2008. Adult-onset amenorrhea: nadotropic hypogonadism and Kallmann’s 23. M. In. The Eating Attitudes Test: al. Bohr Y. Int J Eat Disord thalamic amenorrhea: a controlled com. Girton L. Schoenfeld nology 2008. Disclosure forms provided by the authors are available with gonadism.16:2198. Messager S. except in cases of clear familial in. Hu Y. Bianco SD. All rights reserved. by the National Center for Research Resources (grants 1 UL1 RR025758-01 and M01-RR-01066 to the Harvard thalamic amenorrhea.org. er of gonadotropin-releasing hormone de- pituitary-adrenal axes in athletic women. Chung WC. Metab 1998. and modes of inheritance of gonad- mone pulsatility is disrupted at a thresh. N Engl 21. pin-releasing hormone neurons. The n e w e ng l a n d j o u r na l of m e dic i n e phisms. 14. McArthur JW.3  nejm.19:594. Free alpha-subunit is SS. Seminara SB. Frisch RE. delayed 10. The genetic crine characteristics. crinol 2009. 205. Bishop ME.19: genes in Kallmann syndrome: genotype- 7. 3. J Clin Endocrinol quired for the emergence of gonadotro. Beitins IZ. Moyle SS. and elucidation of the genetic basis of hypothalamic variation in diagnostic criteria. Welt CK. Bullen BA. Tho SP. and Given the limited size of the cohort with hypo. and starvation also contribute to susceptibility Supported by the Eunice Kennedy Shriver National Institute to hypothalamic amenorrhea. syndrome): pathophysiological and ge. Gonadotropin-releasing hormone defi. 20. 2017. hypogonadism. 2007. hypothalamic amenorrhea. EE. 5R01HD015788. Berga SL. The results have been largely inconclu. Hall JE. Crowley WF Jr. et al. Espelage DL. puberty. Bouloux P. et al. Cadman S. and the Pew Latin routinely screened for mutations at loci known American Fellows Program in the Biomedical Sciences (grant to Dr. ology. Turnbull BA. Olmsted MP. heritance of hypothalamic amenorrhea or idio- sive. to underlie idiopathic hypogonadotropic hypo. . J Clin Endocrinol Metab Metab 2003. 1980. Decreased leptin levels in normal 17. Menstrual combinant human leptin in women with peptin and GPR54: discovery of a novel cycles: fatness as a determinant of mini. For personal use only.40:Suppl:S123-S129.org  january 20. nadotropic hypogonadism. 5R01HD42708). Santoro N. of Child Health and Human Development (NICHD) of the Na- In conclusion. psychometric features and clinical corre- 24-hour luteinizing hormone secretory duction of menstrual disorders by strenu. Jr. Anorexia nervosa: immaturity of the Mering G. Pitteloud N. J Neuroendocrinol 2008. Wilfley DE.60:486-92. Thuma JR. satile gonadotropin-releasing hormone. quencies. Mortola JF.org on June 7.291:861-5. The effects of exercise on patterns of neuro-endocrine disturbance females: replication and development of pubertal progression and reproductive as prognostic indicators in hypothalamic a structural model. Beranova M. Katz J. Oliveira LM. Cognitive and J Med 1985. Hayes FJ. Boyar RM. Kaiser UB.88:297-311. Hayes FJ. Loucks AB. Hum Reprod 2001. parison. NICHD-NIH grants 1R01HD056264. Seminara SB. 25. et al. Giles DE. lates. N Engl J Med 2003. 18. J Clin Endocrinol investigation of Kallmann syndrome. and molecular basis of idiopathic hypogo. and phenotype in genes associated with the response to stress are needed. tations in idiopathic hypogonadotropic menstruating women.149:4997-5003. Tsai PS. Martin).20:727-31. Parulekar MS. 5. Fertil Steril 1993. Miller KK. J Clin Endocrinol Metab amenorrhoea. Loucks AB. It might prove amenorrhea and delineation of the relationship more fruitful to investigate whether rare variants among genotype. Classification of eating disor- psychiatric correlates of functional hypo. Aeti. Nat Rev Endo. AU. 2011 The New England Journal of Medicine Downloaded from nejm.51:1150-7. Hypothalamic gonadotrophin. Oliveira LM.86:1532-8. Chan JL. releasing hormone expression in female 22. pattern. ders: toward DSM-V. Schoenfeld D. Skrinar GS. Further heterogeneity in race and ethnic group. 521-39.

Pignatelli D.93:3551-9.org on June 7. nadotropic hypogonadism due to loss of 29. . ceptor 2 and KAL1. Tonra JR.6:706-16. Adult-onset idiopathic 28. Pfaff 863-73. Luteinizing hormone-releasing hor. 27. Dwyer A. Copyright © 2011 Massachusetts Medical Society. the olfactory bulb and reproductive sys. Sidis Y. Mercader JM. Cheng CK. et al. de Roux N. Young J. rodents and monkeys. J Clin Invest 2008. et al. drome and normosmic idiopathic hypo. 2017. Hum Mol Genet 2009. Nachtigall LB. Méndez JP. et al.3  nejm. Bick D. et al. Proc Natl Acad Sci U S A 2006. Gardiner JV. 2003. 31. Plummer L. 103:4140-5. Teixeira L. 22. Monoclonal antibody antagonists of hy. et al. Chung WC. Mol Brain Res 1989.104:17447-52. Proc Natl to KH. DW. Zhang C. Genetic analysis in et al. Falardeau J. Hum Mol Genet A family with hypogonadotropic hypogo. phin-releasing hormone deficiency: molec. with NGF suggest an altered cross-regula- 18:75-81. and their receptors Reversal of idiopathic hypogonadotropic al. 43. Raivio T. 35.” Each article and search result links to this feature.100:10972-6. istence of mutations in prokineticin re. Falardeau J. tem in mice lacking prokineticin receptor function of the KiSS1-derived peptide re- tions in prokineticin 2 and prokineticin PKR2. hypogonadotropic hypogonadism — a al. GnRH-II.336:410-5. Impaired fibroblast growth factor recep. Pearce S. All rights reserved. Decreased FGF8 signaling causes defi. nadal (Kallmann) syndrome. Munguía P. docrinol Metab 2007. Leung PC. tor 1 signaling as a cause of normosmic 37. Prokineticin 2 is a hypothalamic neu. 117:457-63. factor blood levels and gene variability are 4380-90. J Clin Kallmann syndrome: mutations in the Digenic mutations account for variable Endocrinol Metab 2008.6:311-26. Monnier C. Matsu- gonadotropic hypogonadism. 36. et al. Schwanzel-Fukuda M.2(10): tropic hypogonadism. N Engl J versible hypophagia and weight loss in fibroblast growth factor receptor 1 gene. Sidis Y. Dodé C. N Engl J Med 2007. take. Med 1997.org  january 20. n engl j med 364. Muta. Users can create personal folders and move articles into them for convenient retrieval later. PLoS Genet 2006. Söderlund D. Association of NTRK3 and its interaction signalling activity. No other uses without permission. Milgrom E. Abnormal development of da F. ular genetics and clinical spectrum. et (GnRH)-I. Genin E. in humans. 2008. Pitteloud N. For personal use only. Dodé C. et al. pothalamic FGFR1 cause potent but re. Agüera Z. 41. e175. Mercader JM. J Clin Invest 2007. Copyright © 2011 Massachusetts Medical Society. my nejm in the journal online Individual subscribers can store articles and searches using a feature on the Journal’s Web site (NEJM. Altered brain-derived neurotrophic ism. associated with anorexia and bulimia.90:1317. Loss-of-function mutation in the pro.357: ciency of gonadotropin-releasing hormone 34. de Roux N. Boepple PA. idiopathic hypogonadotropic hypogonad. Canto P. Sun HD.292(3):E964-E976. 2011 225 The New England Journal of Medicine Downloaded from nejm. et 45. genes encoding prokineticin-2 and proki. Proc Natl Acad Sci U S A receptor 2 genes in human gonadotro. Diabetes 2010. hypogonadism.30:41-5. Molecular biol. Med 1997. Yamazaki C.17:1234-44. et grate normally in an inherited hypogo. Carel JC. J Androl 2009. Beenken A. 42.org) called “My NEJM. Chaussain JL. Genetic Basis for Hypothalamic Amenorrhea in a single family with a mutation in the pin-releasing hormone receptor. Pralong 118:2822-31. in humans and mice. PROKR2 missense mutations associated 38. Castro JJ. ceptor GPR54. way in eating disorders. Genes Brain Behav 2007. Bataveljic A. Raivio T. mone (LHRH)-expressing cells do not mi. J Clin Endocrinol Metab 2009. Ribasés M. Am J Physiol En- J Clin Endocrinol Metab 2005.26:283-306. 33.337:1597-602. Endocr Rev 2005. et al. Zhang C. Gratacòs M. 44. Malabunga M. Hypogo- Acad Sci U S A 2007. Levilliers J. Fabre L. neticin receptor-2. Matsumoto S. FP. Saus E.94: patients with Kallmann syndrome: coex.59:397-406. nadism and mutations in the gonadotro. with Kallmann syndrome impair receptor al. ogy of gonadotropin-releasing hormone 40. tion of the neurotrophin signaling path- 32. Misrahi M. Pitteloud N. Crowley WF Jr. Patel NA. et al. phenotypes in idiopathic hypogonado- 30. Masumo. ropeptide that potently inhibits food in. Brain Res treatable form of male infertility. et al. Quinton R. N Engl J kineticin 2 gene causes Kallmann syn. 39. Cole LW.