Paul A. Boepple

Digenic mutations account for variable phenotypes in idiopathic hypogonadotropic hypogonadism

Idiopathic hypogonadotropic hypogonadism (IHH) due to defects of gonadotropin-releasing hormone (GnRH) secretion and/or action is a developmental disorder of sexual maturation. To date, several single-gene defects have been implicated in the pathogenesis of IHH. However, significant inter- and intrafamilial variability and apparent incomplete penetrance in familial cases of IHH are difficult to reconcile with the model of a single-gene defect. We therefore hypothesized that mutations at different IHH loci interact in some families to modify their phenotypes. To address this issue, we studied 2 families, one with Kallmann syndrome (IHH and anosmia) and another with normosmic IHH, in which a single-gene defect had been identified: a heterozygous FGF receptor 1 (FGFR1) mutation in pedigree 1 and a compound heterozygous gonadotropin-releasing hormone receptor (GNRHR) mutation in pedigree 2, both of which varied markedly in expressivity within and across families. Further candidate gene screening revealed a second heterozygous deletion in the nasal embryonic LHRH factor (NELF) gene in pedigree 1 and an additional heterozygous FGFR1 mutation in pedigree 2 that accounted for the considerable phenotypic variability. Therefore, 2 different gene defects can synergize to produce a more severe phenotype in IHH families than either alone. This genetic model could account for some phenotypic heterogeneity seen in GnRH deficiency.

Adrenal hypoplasia congenita with hypogonadotropic hypogonadism: evidence that DAX-1 mutations lead to combined hypothalmic and pituitary defects in gonadotropin production.

Adrenal hypoplasia congenita (AHC) is an X-linked disorder that typically presents with adrenal insufficiency during infancy. Hypogonadotropic hypogonadism (HHG) has been identified as a component of this disorder in affected individuals who survive into childhood. Recently, AHC was shown to be caused by mutations in DAX-1, a protein that is structurally similar in its carboxyterminal region to orphan nuclear receptors. We studied two kindreds with clinical features of AHC and HHG. DAX-1 mutations were identified in both families. In the JW kindred, a single base deletion at nucleotide 1219 was accompanied by an additional base substitution that resulted in a frameshift mutation at codon 329 followed by premature termination. In the MH kindred, a GGAT duplication at codon 418 caused a frameshift that also resulted in truncation of DAX-1. Baseline luteinizing hormone (LIT), follicle-stimulating hormone (FSH), and free-alpha-subunit (FAS) levels were determined during 24 h of frequent (q10 min) venous sampling. In patient MH, baseline LH levels were low, but FAS levels were within the normal range. In contrast, in patient JW, the mean LH and FSH were within the normal range during baseline sampling, but LH secretion was erratic rather than showing typical pulses. FAS was apulsatile for much of the day, but a surge was seen over a 3-4-h period. Pulsatile gonadotropin releasing hormone (GnRH) (25 ng/kg) was administered every 2 h for 7 d to assess pituitary responsiveness to exogenous GnRH. MH did not exhibit a gonadotropin response to pulsatile GnRH. JW exhibited a normal response to the first pulse of GnRH, but there was no increase in FAS. In contrast to the priming effect of GnRH in GnRH-deficient patients with Kallmann syndrome, GnRH pulses caused minimal secretory responses of LH and no FAS responses in patient JW. The initial LH response in patient JW implies a deficiency in hypothalamic GnRH. On the other hand, the failure to respond to pulsatile GnRH is consistent with a pituitary defect in gonadotropin production. These two cases exemplify the phenotypic heterogeneity of AHC/HHG, and suggest that DAX-1 mutations impair gonadotropin production by acting at both the hypothalamic and pituitary levels.

Adult-onset idiopathic hypogonadotropic hypogonadism--a treatable form of male infertility.

BACKGROUND Men with isolated gonadotropin-releasing hormone (GnRH) deficiency typically present with an absence of pubertal development. We describe an adult-onset form of idiopathic hypogonadotropic hypogonadism that develops after puberty. METHODS We studied 10 men (age, 27 to 57 years) with normal sexual maturation, idiopathic infertility, sexual dysfunction, low serum testosterone concentrations, and apulsatile secretion of luteinizing hormone on frequent blood sampling. All the men had otherwise normal anterior pituitary hormone secretion and sellar anatomy. We compared the results of semen analyses and measurements of testicular volume, serum testosterone, inhibin B, and gonadotropins in these men with the results in 24 men with classic GnRH deficiency before and during GnRH-replacement therapy and in 29 normal men of similar age. RESULTS Serum gonadotropin concentrations in the men with adult-onset GnRH deficiency were similar before and during pulsatile GnRH administration to those in the men with classic GnRH deficiency. However, as compared with men with classic GnRH deficiency, men with adult-onset hypogonadotropic hypogonadism had larger mean (+/-SD) testicular volumes (18+/-5 vs. 3+/-2 ml, P<0.001), serum testosterone concentrations (78+/-34 vs. 49+/-20 ng per deciliter [2.7+/-1.2 vs. 1.7+/-0.7 nmol per liter], P=0.004), and serum inhibin B concentrations (119+/-52 vs. 60+/-21 pg per milliliter, P<0.001). Treatment with GnRH reversed the hypogonadism and restored fertility in each of the five men who received long-term therapy. CONCLUSIONS The recognition of adult-onset hypogonadotropic hypogonadism in men as a distinct disorder expands the spectrum of GnRH deficiency and identifies a treatable form of male infertility.

Mutations in fibroblast growth factor receptor 1 cause Kallmann syndrome with a wide spectrum of reproductive phenotypes.

BACKGROUND Kallmanns syndrome (KS) is a clinically and genetically heterogeneous disorder consisting of idiopathic hypogonadotropic hypogonadism (IHH) and anosmia. Mutations in KAL1 causing the X-linked form of KS have been identified in 10% of all KS patients and consistently result in a severe reproductive phenotype. KAL1 gene encodes for anosmin-1, a key protein involved in olfactory and GnRH neuronal migration through a putative interaction with FGFR1. Heterozygous mutations in the FGFR1 gene accompanied by a high frequency of cleft palate and other facial dysmorphisms were recently identified in 8% of a large KS cohort, yet the reproductive phenotype of KS patients harboring FGFR1 mutations has not been described. RESULTS One hundred and fifty probands with KS (130 males and 20 females) were studied to determine the frequency and distribution of FGFR1 mutations and their detailed reproductive phenotypes. Fifteen heterozygous mutations in unrelated probands were identified. Twelve missense mutations (p.R78C, p.V102I, p.D224H, p.G237D, p.R254Q, p.V273M, p.E274G, p.Y339C, p.S346C, p.I538V, p.G703S and p.G703R) were distributed among the first, second and third immunoglobulin-like domains (D1-D3), as well as the tyrosine kinase domain (TKD). The mutations Y339C and S346C are located in exon 8B and code for the isoform FGFR1c. Additionally, two nonsense mutations (p.T585X and p.R622X) were documented in the TKD of the protein. A wide spectrum of reproductive function was observed among KS probands including: (1) a severe phenotype demonstrated by microphallus, cryptorchidism, no pubertal development, undetectable serum gonadotropins and low serum testosterone (T) and inhibin B; (2) partial pubertal development; (3) the fertile eunuch variant of IHH with normal testicular size and active spermatogenesis with a reversal of HH after T therapy. In addition, we found an even wider spectrum of reproductive function within pedigrees carrying an FGFR1 mutation ranging from IHH to delayed puberty to normal reproductive function (anosmia only or asymptomatic carriers). These observations strongly suggest a role for other genes that modify the phenotype of FGFR1 mutations. CONCLUSION KS patients and family members carrying an FGFR1 mutation present a broad spectrum of pubertal development in contrast to the almost uniform severe clinical phenotype described in KS subjects with a KAL1 mutation. Additionally, this report implicates the isoform FGFR1c in the pathogenesis of KS.

A Genetic Basis for Functional Hypothalamic Amenorrhea

BACKGROUND Functional hypothalamic amenorrhea is a reversible form of gonadotropin-releasing hormone (GnRH) deficiency commonly triggered by stressors such as excessive exercise, nutritional deficits, or psychological distress. Women vary in their susceptibility to inhibition of the reproductive axis by such stressors, but it is unknown whether this variability reflects a genetic predisposition to hypothalamic amenorrhea. We hypothesized that mutations in genes involved in idiopathic hypogonadotropic hypogonadism, a congenital form of GnRH deficiency, are associated with hypothalamic amenorrhea. METHODS We analyzed the coding sequence of genes associated with idiopathic hypogonadotropic hypogonadism in 55 women with hypothalamic amenorrhea and performed in vitro studies of the identified mutations. RESULTS Six heterozygous mutations were identified in 7 of the 55 patients with hypothalamic amenorrhea: two variants in the fibroblast growth factor receptor 1 gene FGFR1 (G260E and R756H), two in the prokineticin receptor 2 gene PROKR2 (R85H and L173R), one in the GnRH receptor gene GNRHR (R262Q), and one in the Kallmann syndrome 1 sequence gene KAL1 (V371I). No mutations were found in a cohort of 422 controls with normal menstrual cycles. In vitro studies showed that FGFR1 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 mutations may contribute to the variable susceptibility of women to the functional changes in GnRH secretion that characterize hypothalamic amenorrhea. Our observations 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.).

Adrenarche and skeletal maturation during luteinizing hormone releasing hormone analogue suppression of gonadarche.

During puberty the effects of adrenal androgens upon skeletal maturation are obscured by the influence of gonadal steroids. Suppression of gonadarche with an analogue of luteinizing hormone releasing hormone (LHRHa) affords an opportunity to examine the onset and progression of adrenarche in the absence of pubertal levels of gonadal steroids in a controlled fashion and to explore the relationship between adrenal androgens and the rate of epiphyseal maturation. In 29 children with central precocious puberty, gonadarche was suppressed with LHRHa administration for 1-4 yr. During LHRHa exposure, dehydroepiandrosterone sulfate (DHAS) levels, as an index of adrenal maturation, were constant or increased in an age-expected manner. The change in bone age for change in chronologic age decreased from 1.7 +/- 0.1 to 0.49 +/- 0.05 (P = 0.00005), indicating that the LHRHa-induced return to a prepubertal gonadal steroid environment was associated with a slowing of skeletal maturation. DHAS levels were correlated with the rate of skeletal advancement before (r = 0.57, P = 0.001) and during 12 to 48 mo of exposure to LHRHa (r = 0.52, P = 0.003). A negative correlation of DHAS values with subsequent increases in predicted mature height was observed (r = -0.49, P = 0.007). Thus, in children with central precocious puberty, adrenarche progressed normally during LHRHa suppression of gonadarche. In children with the onset of progression of adrenarche during maintenance of a prepubertal gonadal steroid milieu, there was less evidence than in preadrenarchal children of a restraint upon skeletal maturation. These data suggest that adrenal androgens contribute importantly to epiphyseal advancement during childhood.

Trial of Recombinant Follicle-Stimulating Hormone Pretreatment for GnRH-Induced Fertility in Patients with Congenital Hypogonadotropic Hypogonadism

CONTEXT AND OBJECTIVE The optimal strategy for inducing fertility in men with congenital hypogonadotropic hypogonadism (CHH) is equivocal. Albeit a biologically plausible approach, pretreatment with recombinant FSH (rFSH) before GnRH/human chorionic gonadotropin administration has not been sufficiently assessed. The objective of the study was to test this method. DESIGN AND SETTING This was a randomized, open-label treatment protocol at an academic medical center. PATIENTS AND INTERVENTIONS GnRH-deficient men (CHH) with prepubertal testes (<4 mL), no cryptorchidism, and no prior gonadotropin therapy were randomly assigned to either 24 months of pulsatile GnRH therapy alone (inducing endogenous LH and FSH release) or 4 months of rFSH pretreatment followed by 24 months of GnRH therapy. Patients underwent serial testicular biopsies, ultrasound assessments of testicular volume, serum hormone measurements, and seminal fluid analyses. RESULTS rFSH treatment increased inhibin B levels into the normal range (from 29 ± 9 to 107 ± 41 pg/mL, P < .05) and doubled testicular volume (from 1.1 ± 0.2 to 2.2 ± 0.3 mL, P < .005). Histological analysis showed proliferation of both Sertoli cells (SCs) and spermatogonia, a decreased SC to germ cell ratio (from 0.74 to 0.35), and SC cytoskeletal rearrangements. With pulsatile GnRH, the groups had similar hormonal responses and exhibited significant testicular growth. All men receiving rFSH pretreatment developed sperm in their ejaculate (7 of 7 vs 4 of 6 in the GnRH-only group) and showed trends toward higher maximal sperm counts. CONCLUSIONS rFSH pretreatment followed by GnRH is successful in inducing testicular growth and fertility in men with CHH with prepubertal testes. rFSH not only appears to maximize the SC population but also induces morphologic changes, suggesting broader developmental roles.

Relative Roles of Inhibin B and Sex Steroids in the Negative Feedback Regulation of Follicle-Stimulating Hormone in Men across the Full Spectrum of Seminiferous Epithelium Function

CONTEXT AND OBJECTIVE Our aim was to explore the relative roles of gonadal sex steroids and inhibin B in the regulation of FSH across a spectrum of seminiferous epithelium function. SUBJECTS The study included three groups: group I, healthy men (n = 31); group II, men with idiopathic hypogonadotropic hypogonadism receiving pulsatile GnRH (n = 12) selected to represent a spectrum of seminiferous tubular development, testicular size, and baseline inhibin B levels; and group III, men with functional anorchia (n = 3) receiving testosterone replacement. DESIGN Subjects were studied before and after 3 d of acute sex steroid withdrawal. SETTING The study was conducted at the Mallinckrodt General Clinical Research Center of Massachusetts General Hospital. INTERVENTIONS Acute biochemical castration was achieved using high-dose ketoconazole (groups I and II) or withdrawal of androgen therapy (group III). MAIN OUTCOME MEASURES The relationship between FSH and inhibin B in both normal and castrate sex steroid milieu was measured. RESULTS In both normal and castrate sex steroid milieus, there was a negative relationship between inhibin B and FSH, best described by a logarithmic model. Acute biochemical castration resulted in the most dramatic increases in FSH in men with the lowest baseline inhibin B levels. CONCLUSIONS We came to the following conclusions: 1) in the human male, inhibin B is the principal gonadal feedback regulator of FSH secretion unless seminiferous tubular function is severely compromised, and a logarithmic model best describes this relationship; and 2) sex steroid inhibition of FSH secretion is most apparent when serum inhibin B levels fall well below the normal range.

The Relative Role of Gonadal Sex Steroids and Gonadotropin-Releasing Hormone Pulse Frequency in the Regulation of Follicle-Stimulating Hormone Secretion in Men

OBJECTIVE Our objective was to determine the importance of testosterone (T), estradiol (E(2)), and GnRH pulse frequency to FSH regulation in men. DESIGN This was a prospective study with four arms. SETTING The study was performed at the General Clinical Research Center. PATIENTS OR OTHER PARTICIPANTS There were 20 normal (NL) men and 15 men with idiopathic hypogonadotropic hypogonadism (IHH) who completed the study. INTERVENTION Medical castration and inhibition of aromatase were achieved using ketoconazole x 7 d with: 1) no sex steroid addback, 2) T addback starting on d 4, and 3) E(2) addback starting on d 4. IHH men in these arms received GnRH every 120 min. In a further six IHH men receiving ketoconazole with no addback, GnRH frequency was increased to 35 min for d 4-7. Blood was drawn every 10 min x 12 h at baseline, overnight on d 3-4 and 6-7. MAIN OUTCOME MEASURES Mean FSH was calculated from the pool of each frequent sampling study. RESULTS In NL men FSH levels increased from 5.1 +/- 0.7 to 8.7 +/- 1.3 and 9.7 +/- 1.5 IU/liter (P < 0.0001). T caused no suppression of FSH. E(2) reduced FSH from 12.4 +/- 1.8 to 9.3 +/- 1.3 IU/liter (P < 0.05). In IHH men on GnRH every 120 min, FSH levels went from 6.0 +/- 1.6 to 9.0 +/- 3.0 and 11.9 +/- 4.3 (P = 0.07). T caused no suppression of FSH. E(2) decreased FSH such that levels on d 6-7 were similar to baseline. Increasing GnRH frequency to 35 min had no impact on FSH. CONCLUSIONS The sex steroid component of FSH negative feedback in men is mediated by E(2). Increasing GnRH frequency to castrate levels has no impact on FSH in the absence of sex steroids. When inhibin B levels are NL, sex steroids exert a modest effect on FSH.

Gonadotrophin-Releasing Hormone Analogues as Therapeutic Probes in Human Growth and Development: Evidence from Children with Central Precocious Puberty

ABSTRACT. Statural growth and skeletal development were assessed in 87 girls with idiopathic central precocious puberty (CPP) during gonadotrophin‐releasing hormone analogue (GnRHa)‐induced suppression of gonadarche. Before the start of therapy, mean chronological age (CA) was 6.3 years and mean bone age (BA) was 10.6 years. During up to 6 consecutive years of complete suppression of gonadal sex steroid secretion, the mean height velocity decreased from 10.8 cm/year to prepubertal rates. At each interval height velocity was found to be inversely and negatively correlated with BA such that girls with advanced BAs grew at rates well below prepubertal norms but appropriately for their degree of skeletal maturation. Skeletal maturation similarly slowed during prolonged GnRHa administration (ABA/ACA = 0.6 ± 0.1 over 3 years, mean ± SD, n = 66) and was also negatively correlated with the BA before the start of therapy. Predicted adult height increased progressively during therapy; however, when analysed as changes in height SDS(BA), the impact of treatment was variable and correlated positively with the initial degree of skeletal maturation. The effect of GnRHa therapy on growth in children with CPP requires long‐term study and is best analysed by employing a developmental perspective.