LHR Mutations

Inactivating Mutations

In the human male, LH-R function is essential at all ages for testicular androgen production. Therefore, these mutations influence male sexual differentiation, development, and mature functions from the fetal period through adulthood. The inactivating LH-R mutations vary in completeness, and the phenotype of affected males depends on the extent of receptor inactivation (9,59,60). In the mildest forms, the phenotype is hypospadias and/or micropenis, and in the completely inactivating form, the male phe-notype is complete sex reversal, i.e., pseudohermaphroditism (9). The latter group presents with total lack of male type sexual differentiation in utero or postnatal development. The phenotype resembles the complete form of androgen insensitivity syndrome but notably differs by the lack of pubertal breast development, which, in androgen insensitivity, is explained by the increased conversion of testosterone to estradiol. On histological examination, the testes of the individuals with complete LH-R inactivation are totally devoid of Leydig cells, which explains the lack of testosterone production and absent masculinization. However, it is apparent that even on complete LH-R inactivation, some testicular steroidogenesis is possible, because epi-didymides and vasa deferentia are present (9). The explanation for these structures may be low, but functionally sufficient, constitutive steroidogenesis in precursor Leydig cells. This has also been shown with LH-R knockout mice that have a low, but detectable, basal level of steroidogenesis in their testes (61).

In women, LH-R inactivation causes a milder phenotype with normal intrauterine female sex differentiation and a seemingly normal FSH-dependent component of ovarian function (9). Affected women present with primary amenorrhea and normal primary and secondary sex characteristics, increased gonadotropin levels, and a lack of ovarian response to hCG treatment. Histological study of ovarian tissue revealed all stages of follicular development, with the exception of preovulatory follicles and corpora lutea (62). These observations emphasize the importance of LH for ovulation and formation of corpora lutea. LH may also be needed for the maturation of antral to preovulatory follicles (61).

Activating Mutations

The first gonadotropin receptor mutations detected were those activating constitu-tively the LH-R (63,64), perhaps because of their striking phenotype. The syndrome caused by this mutation, i.e., familial male-limited precocious puberty (FMPP), is a gonadotropin-independent form of precocious puberty, also called testotoxicosis. Boys with FMPP begin to exhibit signs and symptoms of puberty between 1 and 3 yr of age. If they remain untreated, puberty progresses rapidly, resulting in premature epiphysial closure and compromised adult height. The syndrome has been know for a long time and was originally assumed to result from a circulating nongonadotropic factor that induces premature LH-independent activation of Leydig cell function. However, the subsequent detection of point mutations in the LH-R gene, which, in transfection studies, caused constitutive activation of the LH signaling pathway in the absence of lig-and, provided a logical molecular pathogenesis of FMPP. Today, more than 10 activating mutations of the LH-R gene are known (see Fig. 2), and they all are localized in or near the transmembrane region of the LHR, which is pivotal for LH signal transduction. The mutations change the conformation of the transmembrane region of the receptor so that it assumes, at least partially, an activated conformation in ligand absence. The consequence is premature activation of Leydig cell testosterone production before the pubertal onset of LH secretion. In cell transfections, the mutated receptor protein usually displays an approx 10-fold increase in basal cAMP production, whereas maximal stimulation is often suppressed. The latter alteration has no physiological importance, because at the circulating levels of LH, receptor occupancy remains relatively low. Whether other signalling pathways that are initiated by LH-R activation are activated to a similar extent has not been systematically studied.

Despite the distinct phenotype in males, no phenotype is known for females with activating LH-R mutation. One explanation is that the LH action initiation in the ovary requires FSH priming, and FSH-dependent paracrine factors may be needed to induce theca cell LH-R responsiveness. Because there is little FSH secretion in these women before the normal age of puberty, the constitutively activated LH-R would not be expected to be prematurely functional. The lack of LH-R in prepubertal granulosa cells is evident, because this receptor is acquired during the later stages of follicular maturation. Therefore, the presence of an activating mutation in the LH-R is functionally unimportant if the gene is not expressed before the age of normal puberty.

A novel phenotype of activating LH-R mutations associated with Leydig cell tumors (LCTs) was described recently in three unrelated boys with isosexual precocious puberty without a family history of this condition (65). Besides the symptoms and findings of FMPP, unilateral testicular mass was found by ultrasonography in all boys. In each case, well-circumscribed tumors were removed that were composed of nested polygonal cells with abundant eosinophilic cytoplasm and round ovoid nuclei. Mitotic activity was low and Reincke's crystals were absent. Genomic DNA was extracted from the tumor tissues and peripheral blood leucocytes, and the sequence-encoding exon 11 of the LH-R was amplified. The tumor tissues, but not leucocytes, contained a heterozygous somatic mutation encoding an Asp578His replacement in LHR (65). When the mutated receptor was transfected into COS-7 cells, it was found to initiate constitutive activation of cAMP production in ligand absence (see Fig. 4). In addition, inositol phosphate production was constitutively activated, a response that has not been made with other activating LH-R mutations. Besides the tumor phenotype, these subjects differ from FMPP because their LH-R mutations were somatic. This particular mutation has not been detected in the germ-line. Two further reports with similar phenotype and causative LH-R mutation have also been published (66,67). It remains to be determined whether this mutation is the only one that causes Leydig cell tumorigenesis.

Another report described nodular LCH in a boy with FMPP, which is not a typical finding in this syndrome (68). The genomic heterozygous Asp564Gly mutation detected in this boy caused only partial activation of cAMP production, and similar Leydig cell alterations were not found in other patients affected with the same mutation. Therefore, the mechanism of the nodular hyperplasia appears to differ from that in the three cases presented here.

A third type of testicular tumor that has occurred in one 36-yr-old patient with FMPP due to a germ-line Asp578Gly mutation is testicular seminoma (69). Although no causative relationship was established for the LH-R mutation and seminoma, the possibility remains that a prolonged high intratesticular testosterone concentration resulting from LH-R stimulation could be oncogenic. However, transgenic (TG) mice that overexpressed hCG and produced high intratesticular testosterone levels showed no signs of testicular tumors beyond mild LCH (S. Rulli, M. Poutanen, & I. Huh-taniemi, unpublished data).

The situation with activating LH-R mutations and testicular tumors is somewhat analogous to familial nonimmunogenic hyperthyroidism and sporadic thyroid adenomas caused by germ-line and somatic mutations of the TSH-R gene, respectively (70,71). As in these thyroid disorders, the somatic LH-R mutations brought about greater constitutive activity of the receptor than do germ-line mutations. The severe forms would probably be eliminated from the genetic pool because of their strong deleterious effects. The mutation detected occurred in a position where three other amino acid-substituting mutations have been detected in Asp578 to Tyr/Glu/Gly (9)

Testosterone Activating Receptors

Fig. 4. Comparison of luteinizing hormone receptor (LH-R) cell signaling by the activating Asp578His or Asp578Tyr mutations and the wild-type (WT) LH-R. Mean basal and human chorionic gonadotropin (hCG)-stimulated accumulation of cAMP (panel A) and inositol phosphates (panel B) is shown in COS-7 cells transfected with DNA for the WT LH-R or receptors with the Asp578Tyr or Asp578His mutation. Basal activity of the WT receptor is the same as that of the pSG5 expression vector alone. (From 65, with permission.)

Fig. 4. Comparison of luteinizing hormone receptor (LH-R) cell signaling by the activating Asp578His or Asp578Tyr mutations and the wild-type (WT) LH-R. Mean basal and human chorionic gonadotropin (hCG)-stimulated accumulation of cAMP (panel A) and inositol phosphates (panel B) is shown in COS-7 cells transfected with DNA for the WT LH-R or receptors with the Asp578Tyr or Asp578His mutation. Basal activity of the WT receptor is the same as that of the pSG5 expression vector alone. (From 65, with permission.)

(see Fig. 2). The other forms cause milder receptor activation and, at most, patchy LCH but no tumors. Hence, the high level of basal stimulation of the receptor is important for adenoma formation. Whether high cAMP production alone or additive or synergistic effect of the concomitantly stimulated phospholipase C pathway are involved in tumorigenesis remains to be established. In fact, the analogous mutation in the TSH-R, Asp633His, has been found in a patient presenting with insular thyroid carcinoma with metastases (72).

These data emphasize that the same phenotype may be caused by both benign FMPP and by LCTs. Therefore long-term follow-up with testicular ultrasonography is advised for boys with FMPP to exclude Leydig cell adenoma. Genetic analysis of the LH-R mutation is also advisable if testicular biopsy material is available. Finally, another genetic cause for LCTs is activating mutation in the stimulatory G protein (73).

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