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The neurokinin B pathway in human reproductionAna Claudia Latronico

studies of rare genetic disorders in humans have yielded important insights into the function of the hypothalamic-pituitary-gonadal axis. a new study now establishes a fundamental role for the neurokinin B pathway in normal reproductive function.

Congenital isolated hypogonatropic hypogo-nadism (IHH) is defined by low gonadal sex steroid levels in the setting of low or normal pituitary secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). It is characterized by complete or par-tial absence of endogenous gonadotropin- releasing hormone (GnRH)-induced gonado-tropin secretion with no anatomical lesion in the hypothalamic-pituitary tract and no other associated pituitary hormone deficiency1. IHH may occur in association with olfactory abnor-malities (Kallmann syndrome), whereas a nor-mal sense of smell defines normosmic IHH.

The pulsatile secretion of GnRH from the hypothalamus is a key requirement for the initiation and maintenance of normal reproductive function2. GnRH release is modulated by excitatory and inhibitory signals generated by neurohormones and neurotransmitters. In the last decade, sev-eral loss-of-function mutations leading to the IHH phenotype have been described in genes encoding different factors implicated in embryonic neuronal migration (anos-min-1, FGF8, FGFR1, PROK2, PROKR2) or in the secretion (kisspeptin, KISS1R) or action (GnRH receptor) of GnRH (Table 1)3–7. However, the genetic basis for IHH has been established in less than 30% of cases, indicating that additional genetic causes could be involved in this condition4. On page 354 of this issue, Topaloglu et al.8 now define mutations in components of the neurokinin B pathway as a new cause of IHH, identify-

ing this pathway as a key regulator of normal human reproductive function.

A new cause of IHHThe tachykinins are a group of structurally related peptides synthesized mainly in the central and peripheral nervous systems9. Substance P, neurokinin A, neurokinin B and hemokinin-1 share a common C-terminal region, characterized by the presence of the Phe-Xaa-Gly-Leu-Met-NH2 motif. In humans, substance P is the most extensively studied tachykinin owing to its involvement in several inflammatory and immune cells10. Topaloglu et al.8 identified a critical role for neurokinin B in the human reproductive axis by using genome-wide SNP analysis in nine inbred Turkish families with idiopathic

IHH. They found rare missense mutations in TAC3 or TACR3, encoding neurokinin B and its receptor NKR3, respectively, in four of these families with IHH. In three families, the authors found homozygous mutations (encoding G93D and P353S) in the trans-membrane domain of NKR3, and in one fam-ily, they found a M90T substitution located at the canonical tachykinin motif of mature neurokinin B. Segregation analysis revealed that heterozygous carriers of the M90T substi-tution were unaffected, indicating autosomal recessive IHH. In vitro analysis showed that both ligand and receptor mutations resulted in impaired receptor signaling in a heterolo-gous cell line. Notably, all individuals with homozygous TAC3 or TACR3 mutations showed a severe gonadotropin deficiency

Ana Claudia Latronico is in the Developmental Endocrinology Unit, Hormone and Molecular Genetic Laboratory LIM/42, Clinical Hospital, Sao Paulo Medical School, University of Sao Paulo, Sao Paulo 05403-900, Brazil. e-mail: anacl@usp.br

nature genetics | volume 41 | number 3 | march 2009 269

Table 1 Mutations associated with congenital isolated hypogonadotropic hypogonadism in humansGene Locus Gene product Function in the

reproductive axisInheritance Phenotype

KAL1 Xp22.3 Anosmin-1 Migration of GnRH neurons

X-linked Kallmann syndrome

FGF8,

FGFR1

10q25

8p12

Fibroblast growth factor-8 and its receptor 1

Migration of GnRH neurons

Autosomal dominant

Kallmann syndrome or normosmic IHH

NELF 9q34.3 Nasal embryonic LHRH factor

Migration of GnRH neurons

Autosomal dominant?

Kallmann syndrome

PROK2,

PROKR2

3p21.1,

20p12.3

Prokineticin-2 and its receptor

Migration of GnRH neurons

Autosomal recessive

Kallmann syndrome or normosmic IHH

GNRHR 4q21.2 GnRH receptor GnRH signalling Autosomal recessive

Normosmic IHH

KISS1R 19p13.3 KISS1 receptor Stimulation of GnRH secretion

Autosomal recessive

Normosmic IHH

NR0B1 Xp21 Orphan nuclear receptor

Regulation of pituitary and hypothalamic gene transcription

X-linked Adrenal insufficiency and normosmic IHH

LEPR 1p31 Leptin receptor GnRH modulation Autosomal recessive

Severe obesity and normosmic IHH

TAC3,

TACR3

12q13–12,

4q25

Neurokinin B and its receptor

Unknown Autosomal recessive

Normosmic IHH

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without olfactory abnormalities or other additional nonreproductive anomalies.

To date, TACR3 mutations seem to be an uncommon cause of familial or sporadic IHH, as the DNA analysis of six other families and 50 individuals from a variety of ancestry groups with IHH did not reveal additional mutations. Similarly, loss-of-function muta-tions in KISS1R (also known as GPR54) are considered a rare cause of GnRH deficiency in humans, despite the fundamental impor-tance of the kisspeptin–KISS1R pathway in modulating GnRH secretion3. However, an extensive analysis of TAC3 and TACR3 in addi-tional populations will be needed to determine the true mutation frequency and the possible occurrence of milder phenotypes.

Next challengesThe mechanism whereby the neurokinin B system exerts its effects on the central neu-roendocrine control of human reproduction remains unknown. Nevertheless, several lines of evidence suggest that neurokinin B might have a role as a regulator of GnRH secretion. Neurokinin B expression in the rat median eminence of the hypothalamus, as well as the expression of its receptor on GnRH neurons, argues that this system may directly modulate GnRH neuron function11. Studies in both human and nonhuman primates showed that

gonadotropin hypersecretion in postmeno-pausal females was secondary to increased GnRH secretion in association with hypertro-phy of neurons expressing KISS1, neurokinin B, substance P, dynorphin and estrogen recep-tor α, suggesting that neurokinin B is part of the neural network responsible for gonado-tropin release12. Physiological and pharmaco-logical studies conducted in different animal models, especially in primates, will be neces-sary to demonstrate the precise mechanistic role of the neurokinin B pathway in the repro-ductive axis. It would be interesting to further investigate the effects of neurokinin B admin-istration, either centrally or peripherally, on LH and FSH secretion in vivo. Finally, the characterization of reproductive phenotypes of mice lacking Tac2 (the murine ortholog of TAC3) or Tacr3 remains to be explored.

IHH is a clinically and genetically heteroge-neous disease. Digenic mutations have been reported to account for variable phenotypes in familial IHH13. It is possible that TAC3 or TACR3 mutations can synergize with other genetic defects to produce phenotypic het-erogeneity in IHH families. Notably, a gain-of-function mutation (encoding R386P) in KISS1R (GPR54) was recently reported in a girl with idiopathic central precocious puberty, the converse phenotype of IHH14. In vitro, the R386P substitution leads to prolonged activa-

tion of the intracellular signaling pathway in response to kisspeptin. Therefore, it is also rea-sonable to hypothesize that gain-of-function mutation in TACR3, encoding a G-protein coupled receptor like KISS1R, might be identi-fied in children with gonadotropin-dependent precocious puberty. The discovery of TAC3 and TACR3 mutations in familial IHH has left numerous challenges for continuing studies of the neurokinin B system in the reproductive axis, including the establishment of its precise biological role in GnRH secretion.

1. Trarbach, E.B. et al. Pituitary 10, 381–391 (2007).2. Grumbach, M.M. Horm. Res. 57, 2–14 (2002).3. Seminara, S.B. & Crowley, W.F., Jr. J. Neuroendocrinol.

20, 727–731 (2008).4. Dodé, C. & Hardelin, J.P. Eur. J. Hum. Genet. 17,

139–146 (2009).5. Hardelin, J.P. & Dodé, C. Sex. Dev. 2, 181–193

(2008).6. Pitteloud, N. et al. Proc. Natl. Acad. Sci. USA 103,

6281–6286 (2006).7. Pitteloud, N. et al. Proc. Natl. Acad. Sci. USA 104,

17447–17452 (2007).8. Topaloglu, A.K. et al. Nat. Genet. 41, 354–358

(2009). 9. Almeida, T.A. et al. Curr. Med. Chem. 11, 2045–2081

(2004).10. Klassert, T.E. et al. J. Neuroimmunol. 196, 27–34

(2008).11. Krajewski, S.J. et al. J. Comp. Neurol. 489, 372–386

(2005).12. Rance, N.E. Peptides 30, 111–122 (2009).13. Pitteloud, N. et al. J. Clin. Invest. 117, 457–463

(2007).14. Teles, M.G. et al. N. Engl. J. Med. 358, 709–715

(2008).

270 volume 41 | number 3 | march 2009 | nature genetics

Processing the H3K36me3 signatureRobert J Sims III & Danny Reinberg

The global patterning of histone lysine methylation has been scrutinized over the years in an effort to uncover unique features indicative of chromatin function. a study in Caenorhabditis elegans now shows that nucleosomes covering exons and introns on active genes are differentially marked by H3K36 trimethylation, suggesting a new mode of communication between chromatin and pre-mRna processing.

Robert J. Sims III is at Constellation Pharmaceuticals, Cambridge, Massachusetts 02139, USA. Danny Reinberg is at the Department of Biochemistry, New York University School of Medicine, New York 10016, USA. e-mail: reinbd01@nyumc.org or robert.sims@constellationpharma.com

Chromatin has a prominent role in the regu-lation of cellular processes that require DNA access, such as transcription and DNA repli-cation and repair. Post-translational, covalent modifications (PTMs) of histones provide one mechanism to differentiate otherwise identical chromatin segments for distinct

functional purposes. Although advancements have been made in cataloguing the wide vari-ety of PTMs on histones on a genome-wide scale, understanding how these patterns functionally contribute to specific biologi-cal processes remains a major challenge. On page 376 of this issue, Julie Ahringer and col-leagues1 have identified a specific chromatin signature in C. elegans that demarks expressed exons on actively transcribed genes, revealing a potential mechanism to direct splicing.

Mining for modificationsHistone lysine methylation has garnered spe-cial attention in part because of its impor-tance in the maintenance of chromatin

architecture and in long-term patterns of gene expression and its potential involvement in epigenetic phenomena2. Moreover, lysine methylation has inherent indexing potential because it exists in four flavors, unmodified, mono-, di- and trimethylation, each of which yield distinct biological consequences. To better understand the chromatin landscape of the model organism C. elegans, Kolasinska-Zwierz et al.1 assessed the genome-wide location of three well-defined histone modi-fications, trimethylation of lysine 4, 9 and 36 of histone H3 (H3K4me3, H3K9me3 and H3K36me3). Consistent with studies in yeast, flies and mammals, H3K4me3 and H3K36me3 were found tightly associated

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