POU6F2 mutation in humans with pubertal failure alters GnRH transcript expression.

Idiopathic hypogonadotropic hypogonadism (IHH) is characterized by the absence of pubertal development and subsequent impaired fertility often due to gonadotropin-releasing hormone (GnRH) deficits. Exome sequencing of two independent cohorts of IHH patients identified 12 rare missense variants in POU6F2 in 15 patients. POU6F2 encodes two distinct isoforms. In the adult mouse, expression of both isoform1 and isoform2 was detected in the brain, pituitary, and gonads. However, only isoform1 was detected in mouse primary GnRH cells and three immortalized GnRH cell lines, two mouse and one human. To date, the function of isoform2 has been verified as a transcription factor, while the function of isoform1 has been unknown. In the present report, bioinformatics and cell assays on a human-derived GnRH cell line reveal a novel function for isoform1, demonstrating it can act as a transcriptional regulator, decreasing GNRH1 expression. In addition, the impact of the two most prevalent POU6F2 variants, identified in five IHH patients, that were located at/or close to the DNA-binding domain was examined. Notably, one of these mutations prevented the repression of GnRH transcripts by isoform1. Normally, GnRH transcription increases as GnRH cells mature as they near migrate into the brain. Augmentation earlier during development can disrupt normal GnRH cell migration, consistent with some POU6F2 variants contributing to the IHH pathogenesis.

PLXNB1 mutations in the etiology of idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) comprises a group of rare genetic disorders characterized by pubertal failure caused by gonadotropin-releasing hormone (GnRH) deficiency. Genetic factors involved in semaphorin/plexin signaling have been identified in patients with IHH. PlexinB1, a member of the plexin family receptors, serves as the receptor for semaphorin 4D (Sema4D). In mice, perturbations in Sema4D/PlexinB1 signaling leads to improper GnRH development, highlighting the importance of investigating PlexinB1 mutations in IHH families. In total, 336 IHH patients (normosmic IHH, n = 293 and Kallmann syndrome, n = 43) from 290 independent families were included in the present study. Six PLXNB1 rare sequence variants (p.N361S, p.V608A, p.R636C, p.V672A, p.R1031H, and p.C1318R) are described in eight normosmic IHH patients from seven independent families. These variants were examined using bioinformatic modeling and compared to mutants reported in PLXNA1. Based on these analyses, the variant p.R1031H was assayed for alterations in cell morphology, PlexinB1 expression, and migration using a GnRH cell line and Boyden chambers. Experiments showed reduced membrane expression and impaired migration in cells expressing this variant compared to the wild-type. Our results provide clinical, genetic, molecular/cellular, and modeling evidence to implicate variants in PLXNB1 in the etiology of IHH.

Inactivating NHLH2 variants cause idiopathic hypogonadotropic hypogonadism and obesity in humans.

Metabolism has a role in determining the time of pubertal development and fertility. Nonetheless, molecular/cellular pathways linking metabolism/body weight to puberty/reproduction are unknown. The KNDy (Kisspeptin/Neurokinin B/Dynorphin) neurons in the arcuate nucleus of the hypothalamus constitute the GnRH (gonadotropin-releasing hormone) pulse generator. We previously created a mouse model with a whole-body targeted deletion of nescient helix-loop-helix 2 (Nhlh2; N2KO), a class II member of the basic helix-loop-helix family of transcription factors. As this mouse model features pubertal failure and late-onset obesity, we wanted to study whether NHLH2 represents a candidate molecule to link metabolism and puberty in the hypothalamus. Exome sequencing of a large Idiopathic Hypogonadotropic Hypogonadism cohort revealed obese patients with rare sequence variants in NHLH2, which were characterized by in-silico protein analysis, chromatin immunoprecipitation, and luciferase reporter assays. In vitro heterologous expression studies demonstrated that the variant p.R79C impairs Nhlh2 binding to the Mc4r promoter. Furthermore, p.R79C and other variants show impaired transactivation of the human KISS1 promoter. These are the first inactivating human variants that support NHLH2's critical role in human puberty and body weight control. Failure to carry out this function results in the absence of pubertal development and late-onset obesity in humans.

Review of human genetic and clinical studies directly relevant to GnRH signalling.

GnRH is the pivotal hormone in controlling the hypothalamic-pituitary gonadal (HPG) axis in humans and other mammalian species. GnRH function is influenced by a multitude of known and still unknown environmental and genetic factors. Molecular genetic studies on human families with hypogonadotropic hypogonadism over the past two decades have been instrumental in delineating the kisspeptin and neurokinin B signalling, which integrally modulates GnRH release from the hypothalamus. The identification of kisspeptin and neurokinin B ligand-receptor gene pair mutations in patients with absent puberty have paved the way to a greater understanding of the central regulation of the HPG cascade. In this article, we aim to review the literature on the genetic and clinical aspects of GnRH and its receptor, as well as the two ligand-receptor sets directly pertinent to the function of GnRH hormone signalling, kisspeptin/ kisspeptin receptor and NKB/NK3R.

Loss-of-function variants in SEMA3F and PLXNA3 encoding semaphorin-3F and its receptor plexin-A3 respectively cause idiopathic hypogonadotropic hypogonadism.

PURPOSE: Idiopathic hypogonadotropic hypogonadism (IHH) is characterized by absent puberty and subsequent infertility due to gonadotropin-releasing hormone (GnRH) deficiency. IHH can be accompanied by normal or compromised olfaction (Kallmann syndrome). Several semaphorins are known potent modulators of GnRH, olfactory, and vomeronasal system development. In this study, we investigated the role of Semaphorin-3F signaling in the etiology of IHH. METHODS: We screened 216 IHH patients by exome sequencing. We transiently transfected HEK293T cells with plasmids encoding wild type (WT) or corresponding variants to investigate the functional consequences. We performed fluorescent IHC to assess SEMA3F and PLXNA3 expression both in the nasal region and at the nasal/forebrain junction during the early human fetal development. RESULTS: We identified ten rare missense variants in SEMA3F and PLXNA3 in 15 patients from 11 independent families. Most of these variants were predicted to be deleterious by functional assays. SEMA3F and PLXNA3 are both expressed along the olfactory nerve and intracranial projection of the vomeronasal nerve/terminal nerve. PLXNA1-A3 are expressed in the early migratory GnRH neurons. CONCLUSION: SEMA3F signaling through PLXNA1-A3 is involved in the guidance of GnRH neurons and of olfactory and vomeronasal nerve fibers in humans. Overall, our findings suggest that Semaphorin-3F signaling insufficiency contributes to the pathogenesis of IHH.

Prevalence and associated phenotypes of PLXNA1 variants in normosmic and anosmic idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) can be divided into two major forms, normosmic IHH and Kallmann syndrome (KS). Genetic mutations are responsible for the majority of IHH. PLXNA1 has recently been implicated in the GnRH neuron migration and the etiology of KS. We aimed to investigate the prevalence and associated phenotypes of PLXNA1 variants in a large cohort of IHH patients. We screened the whole exome data of 215 IHH patients in a single center for causative PLXNA1 variants. Our studies showed eight novel (p.Arg836His, p.Lys1451Arg, p.Val287Met, p.Val536Ile, p.Ser1850Arg, p.Ile1701Val, p.Arg319Trp, and p.Pro485Leu) and two previously described (p.Arg528Trp and p.Gly720Glu) heterozygous PLXNA1 variants in nine affected individuals from seven unrelated families. Only three of nine patients were anosmic (KS) while the remaining patients showed normal olfactory function (nIHH). Seven of nine patients (77.7%) harbored additional one or two variants in other nIHH/KS-associated genes, including PROKR2, IGSF10, HS6ST1, SEMA3E, CCDC141, FGFR1, NRP1, POLR3A, and SRA1. Our findings indicate that PLXNA1 variants cause not only anosmic but also normosmic IHH with a relatively high prevalence (3.9%). Heterozygous missense PLXNA1 variants appear to be involved together with other IHH gene variants in bringing about the IHH disease phenotype.

Update on the Genetics of Idiopathic Hypogonadotropic Hypogonadism.

Traditionally, idiopathic hypogonadotropic hypogonadism (IHH) is divided into two major categories: Kallmann syndrome (KS) and normosmic IHH (nIHH). To date, inactivating variants in more than 50 genes have been reported to cause IHH. These mutations are estimated to account for up to 50% of all apparently hereditary cases. Identification of further causative gene mutations is expected to be more feasible with the increasing use of whole exome/genome sequencing. Presence of more than one IHH-associated mutant gene in a given patient/pedigree (oligogenic inheritance) is seen in 10-20% of all IHH cases. It is now well established that about 10-20% of IHH cases recover from IHH either spontaneously or after receiving some sex steroid replacement therapy. Moreover, there may be an overlap or transition between constitutional delay in growth and puberty (CDGP) and IHH. It has been increasingly observed that oligogenic inheritance and clinical recovery complicates the phenotype/genotype relationship in IHH, thus making it challenging to find new IHH-associated genes. In a clinical sense, recognizing those IHH genes and associated phenotypes may improve our diagnostic capabilities by enabling us to prioritize the screening of particular gene(s) such as synkinesia (ANOS1), dental agenesis (FGF8/FGFR1) and hearing loss (CHD7). Also, IHH-associated gene studies may be translated into new therapies such as for polycystic ovary syndrome. In a scientific sense, the most significant contribution of IHH-associated gene studies has been the characterization of the long-sought gonadotropin releasing hormone pulse generator. It appears that genetic studies of IHH will continue to advance our knowledge in both the biological and clinical domains.

CCDC141 Mutations in Idiopathic Hypogonadotropic Hypogonadism.

Context: Gonadotropin-releasing hormone neurons originate outside the central nervous system in the olfactory placode and migrate into the central nervous system, becoming integral components of the hypothalamic-pituitary-gonadal axis. Failure of this migration can lead to idiopathic hypogonadotropic hypogonadism (IHH)/Kallmann syndrome (KS). We have previously shown that CCDC141 knockdown leads to impaired migration of GnRH neurons but not of olfactory receptor neurons. Objective: The aim of this study was to further describe the phenotype and prevalence of CCDC141 mutations in IHH/KS. Design: Using autozygosity mapping, candidate gene screening, whole-exome sequencing, and Sanger sequencing, those individuals carrying deleterious CDCD141 variants and their phenotypes were determined in a cohort of 120 IHH/KS families. Patients and Interventions: No interventions were made. Results: Our studies revealed nine affected individuals from four independent families in which IHH/KS is associated with inactivating CCDC141 variants, revealing a prevalence of 3.3%. Affected individuals (with the exception of those from family 1 who concomitantly have FEZF1 mutations) have normal olfactory function and anatomically normal olfactory bulbs. Four affected individuals show evidence of clinical reversibility. In three of the families, there was at least one more potentially deleterious variant in other known puberty genes with evidence of allelic heterogeneity within respective pedigrees. Conclusions: These studies confirm that inactivating CCDC141 variants cause normosmic IHH but not KS. This is consistent with our previous in vitro experiments showing exclusively impaired embryonic migration of GnRH neurons upon CCDC141 knockdown. These studies expand the clinical and genetic spectrum of IHH and also attest to the complexity of phenotype and genotype in IHH.

Hypogonadotropic Hypogonadism due to Novel FGFR1 Mutations.

OBJECTIVE: The underlying genetic etiology of hypogonadotropic hypogonadism (HH) is heterogeneous. Fibroblast growth factor signaling is pivotal in the ontogeny of gonadotropin-releasing hormone neurons. Loss-of-function mutations in FGFR1 gene cause variable HH phenotypes encompassing pubertal delay to idiopathic HH (IHH) or Kallmann syndrome (KS). As FGFR1 mutations are common, recognizing mutations and associated phenotypes may enhance clinical management. METHODS: Using a candidate gene approach, we screened 52 IHH/KS patients. RESULTS: We identified three novel (IVS3-1G>C and p.W2X, p.R209C) FGFR1 gene mutations. Despite predictive null protein function, patients from the novel mutation families had normosmic IHH without non-reproductive phenotype. CONCLUSION: These findings further emphasize the great variability of FGFR1 mutation phenotypes in IHH/KS.

Idiopathic Hypogonadotropic Hypogonadism Caused by Inactivating Mutations in SRA1.

OBJECTIVE: What initiates the pubertal process in humans and other mammals is still unknown. We hypothesized that gene(s) taking roles in triggering human puberty may be identified by studying a cohort of idiopathic hypogonadotropic hypogonadism (IHH). METHODS: A cohort of IHH cases was studied based on autozygosity mapping coupled with whole exome sequencing. RESULTS: Our studies revealed three independent families in which IHH/delayed puberty is associated with inactivating SRA1 variants. SRA1 was the first gene to be identified to function through its protein as well as noncoding functional ribonucleic acid products. These products act as co-regulators of nuclear receptors including sex steroid receptors as well as SF-1 and LRH-1, the master regulators of steroidogenesis. Functional studies with a mutant SRA1 construct showed a reduced co-activation of ligand-dependent activity of the estrogen receptor alpha, as assessed by luciferase reporter assay in HeLa cells. CONCLUSION: Our findings strongly suggest that SRA1 gene function is required for initiation of puberty in humans. Furthermore, SRA1 with its alternative products and functionality may provide a potential explanation for the versatility and complexity of the pubertal process.

CCDC141 Mutation Identified in Anosmic Hypogonadotropic Hypogonadism (Kallmann Syndrome) Alters GnRH Neuronal Migration.

The first mutation in a gene associated with a neuronal migration disorder was identified in patients with Kallmann Syndrome, characterized by hypogonadotropic hypogonadism and anosmia. This pathophysiological association results from a defect in the development of the GnRH and the olfactory system. A recent genetic screening of Kallmann Syndrome patients revealed a novel mutation in CCDC141. Little is known about CCDC141, which encodes a coiled-coil domain containing protein. Here, we show that Ccdc141 is expressed in GnRH neurons and olfactory fibers and that knockdown of Ccdc141 reduces GnRH neuronal migration. Our findings in human patients and mouse models predict that CCDC141 takes part in embryonic migration of GnRH neurons enabling them to form a hypothalamic neuronal network to initiate pulsatile GnRH secretion and reproductive function.

Genetics of Hypogonadotropic Hypogonadism.

Hypogonadotropic hypogonadism (HH) often manifests as pubertal delay. A considerable proportion of cases of HH is due to genetic mutations. Recognizing those mutated genes and associated phenotypes may improve our diagnostic capabilities. GNRHR and TACR3 should be the first two genes to be screened in a clinical setting for equivocal cases such as constitutional delay in puberty versus idiopathic HH. In Kallmann syndrome (KS), according to the presence of certain accompanying clinical features, genetic screening for particular gene(s) may be prioritized: synkinesia (KAL1), dental agenesis (FGF8/FGFR1), bony anomalies (FGF8/FGFR1), and hearing loss (CHD7, SOX10). FEZF1 has recently been added to the growing list of KS genes. Also, discovery of mutations in KISS1/KISS1R and TAC3/TACR3 in kisspeptin and neurokinin B signaling, respectively, has provided major advancements in our understanding of the biology of the gonadotropin-releasing hormone pulse generator. Identification of further causative mutations accounting for the HH phenotype, which is now more feasible with the increasing popularity of whole exome sequencing, may provide deeper insight into the biology of the hypothalamic-pituitary-gonadal axis.

Complete Idiopathic Hypogonadotropic Hypogonadism due to Homozygous GNRH1 Mutations in the Mutational Hot Spots in the Region Encoding the Decapeptide.

INTRODUCTION: Mutations of the human GNRH1 gene are an extremely rare cause of normosmic idiopathic hypogonadotropic hypogonadism (nIHH), with only 6 mutations so far described. PATIENTS: As part of a larger study, families with IHH were screened for mutations in genes known to be associated with IHH. In family 1, a 15-year and 9-month-old boy first presented during infancy with micropenis and bilateral cryptorchidism. His pubic and axillary hair is at stage 4 and 2, respectively. His testes are 1 ml bilaterally, and his stretched penile length is 3.6 cm. In family 2, a 19-year and 2-month-old man was referred because of absence of secondary sexual characteristics. His 13-year and 8-month-old sister did not have any breast development. RESULTS: In 3 patients from 2 independent families we identified GNRH1 mutations. In the proband from family 1, a homozygous 1-base deletion (c.87delA) leading to a frameshift mutation (p.G29GfsX12) was identified. In family 2, the affected siblings had a novel homozygous mutation of c.G92A leading to p.R31H. CONCLUSION: Both mutations in these families are located in the region encoding the decapeptide and in the loci where the mutations have been described before. Therefore, these areas can be considered as mutational hot spots, indicating priority for routine diagnostic gene mutation analysis.

Normosmic idiopathic hypogonadotropic hypogonadism due to a novel homozygous nonsense c.C969A (p.Y323X) mutation in the KISS1R gene in three unrelated families.

OBJECTIVE: The spectrum of genetic alterations in cases of hypogonadotropic hypogonadism continue to expand. However, KISS1R mutations remain rare. The aim of this study was to understand the molecular basis of normosmic idiopathic hypogonadotropic hypogonadism. METHODS: Clinical characteristics, hormonal studies and genetic analyses of seven cases with idiopathic normosmic hypogonadotropic hypogonadism (nIHH) from three unrelated consanguineous families are presented. RESULTS: One male presented with absence of pubertal onset and required surgery for severe penoscrotal hypospadias and cryptorchidism, while other two males had absence of pubertal onset. Two of four female cases required replacement therapy for pubertal onset and maintenance, whereas the other two had spontaneous pubertal onset but incomplete maturation. In sequence analysis, we identified a novel homozygous nonsense (p.Y323X) mutation (c.C969A) in the last exon of the KISS1R gene in all clinically affected cases. CONCLUSIONS: We identified a homozygous nonsense mutation in the KISS1R gene in three unrelated families with nIHH, which enabled us to observe the phenotypic consequences of this rare condition. Escape from nonsense-mediated decay, and thus production of abnormal proteins, may account for the variable severity of the phenotype. Although KISS1R mutations are extremely rare and can cause a heterogeneous phenotype, analysis of the KISS1R gene should be a part of genetic analysis of patients with nIHH, to allow better understanding of phenotype-genotype relationship of KISS1R mutations and the underlying genetic basis of patients with nIHH.

MCM9 mutations are associated with ovarian failure, short stature, and chromosomal instability.

Premature ovarian failure (POF) is genetically heterogeneous and manifests as hypergonadotropic hypogonadism either as part of a syndrome or in isolation. We studied two unrelated consanguineous families with daughters exhibiting primary amenorrhea, short stature, and a 46,XX karyotype. A combination of SNP arrays, comparative genomic hybridization arrays, and whole-exome sequencing analyses identified homozygous pathogenic variants in MCM9, a gene implicated in homologous recombination and repair of double-stranded DNA breaks. In one family, the MCM9 c.1732+2T>C variant alters a splice donor site, resulting in abnormal alternative splicing and truncated forms of MCM9 that are unable to be recruited to sites of DNA damage. In the second family, MCM9 c.394C>T (p.Arg132(∗)) results in a predicted loss of functional MCM9. Repair of chromosome breaks was impaired in lymphocytes from affected, but not unaffected, females in both families, consistent with MCM9 function in homologous recombination. Autosomal-recessive variants in MCM9 cause a genomic-instability syndrome associated with hypergonadotropic hypogonadism and short stature. Preferential sensitivity of germline meiosis to MCM9 functional deficiency and compromised DNA repair in the somatic component most likely account for the ovarian failure and short stature.

Mutations in FEZF1 cause Kallmann syndrome.

Gonadotropin-releasing hormone (GnRH) neurons originate outside the CNS in the olfactory placode and migrate into the CNS, where they become integral components of the hypothalamic-pituitary-gonadal (HPG) axis. Disruption of this migration results in Kallmann syndrome (KS), which is characterized by anosmia and pubertal failure due to hypogonadotropic hypogonadism. Using candidate-gene screening, autozygosity mapping, and whole-exome sequencing in a cohort of 30 individuals with KS, we searched for genes newly associated with KS. We identified homozygous loss-of-function mutations in FEZF1 in two independent consanguineous families each with two affected siblings. The FEZF1 product is known to enable axons of olfactory receptor neurons (ORNs) to penetrate the CNS basal lamina in mice. Because a subset of axons in these tracks is the migratory pathway for GnRH neurons, in FEZF1 deficiency, GnRH neurons also fail to enter the brain. These results indicate that FEZF1 is required for establishment of the central component of the HPG axis in humans.

Loss-of-function mutations in PNPLA6 encoding neuropathy target esterase underlie pubertal failure and neurological deficits in Gordon Holmes syndrome.

CONTEXT: Gordon Holmes syndrome (GHS) is characterized by cerebellar ataxia/atrophy and normosmic hypogonadotropic hypogonadism (nHH). The underlying pathophysiology of this combined neurodegeneration and nHH remains unknown. OBJECTIVE: We aimed to provide insight into the disease mechanism in GHS. METHODS: We studied a cohort of 6 multiplex families with GHS through autozygosity mapping and whole-exome sequencing. RESULTS: We identified 6 patients from 3 independent families carrying loss-of-function mutations in PNPLA6, which encodes neuropathy target esterase (NTE), a lysophospholipase that maintains intracellular phospholipid homeostasis by converting lysophosphatidylcholine to glycerophosphocholine. Wild-type PNPLA6, but not PNPLA6 bearing these mutations, rescued a well-established Drosophila neurodegenerative phenotype caused by the absence of sws, the fly ortholog of mammalian PNPLA6. Inhibition of NTE activity in the LßT2 gonadotrope cell line diminished LH response to GnRH by reducing GnRH-stimulated LH exocytosis, without affecting GnRH receptor signaling or LHß synthesis. CONCLUSION: These results suggest that NTE-dependent alteration of phospholipid homeostasis in GHS causes both neurodegeneration and impaired LH release from pituitary gonadotropes, leading to nHH.

A rare variant in human fibroblast activation protein associated with ER stress, loss of enzymatic function and loss of cell surface localisation.

Fibroblast activation protein (FAP) is a focus of interest as a potential cancer therapy target. This membrane bound protease possesses the unique catalytic activity of hydrolysis of the post-proline bond two or more residues from the N-terminus of substrates. FAP is highly expressed in activated fibroblastic cells in tumours, arthritis and fibrosis. A rare, novel, human polymorphism, C1088T, encoding Ser363 to Leu, occurring in the sixth blade of the ß propeller domain, was identified in a family. Both in primary human fibroblasts and in Ser363LeuFAP transfected cells, we showed that this single substitution ablates FAP dimerisation and causes loss of enzyme activity. Ser363LeuFAP was detectable only in endoplasmic reticulum (ER), in contrast to the distribution of wild-type FAP on the cell surface. The variant FAP showed decreased conformational antibody binding, consistent with an altered tertiary structure. Ser363LeuFAP expression was associated with upregulation of the ER chaperone BiP/GRP78, ER stress sensor ATF6, and the ER stress response target phospho-eIF2α, all indicators of ER stress. Proteasomal inhibition resulted in accumulation of Ser363LeuFAP, indicating the involvement of ER associated degradation (ERAD). Neither CHOP expression nor apoptosis was elevated, so ERAD is probably important for protecting Ser363LeuFAP expressing cells. These data on the first loss of function human FAP gene variant indicates that although the protein is vulnerable to an amino acid substitution in the ß-propeller domain, inactive, unfolded FAP can be tolerated by cells.

Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs.

The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.

Inactivating KISS1 mutation and hypogonadotropic hypogonadism.

Gonadotropin-releasing hormone (GnRH) is the central regulator of gonadotropins, which stimulate gonadal function. Hypothalamic neurons that produce kisspeptin and neurokinin B stimulate GnRH release. Inactivating mutations in the genes encoding the human kisspeptin receptor (KISS1R, formerly called GPR54), neurokinin B (TAC3), and the neurokinin B receptor (TACR3) result in pubertal failure. However, human kisspeptin loss-of-function mutations have not been described, and contradictory findings have been reported in Kiss1-knockout mice. We describe an inactivating mutation in KISS1 in a large consanguineous family that results in failure of pubertal progression, indicating that functional kisspeptin is important for puberty and reproduction in humans. (Funded by the Scientific and Technological Research Council of Turkey [TÜBITAK] and others.).