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Male Pseudohermaphroditism Due to Inactivating Luteinizing Hormone Receptor Mutations
Archives of Medical Research 30 (1999) 495–500
REVIEW ARTICLE
Male Pseudohermaphroditism Due to Inactivating Luteinizing Hormone Receptor Mutations Shao-Ming Wu* and Wai-Yee Chan**,*** Departments of *Pediatrics, **Biochemistry & Molecular Biology, and ***Cell Biology, Georgetown University, Washington, DC, USA Received for publication September 9, 1999; accepted September 9, 1999 (99/159).
Human male sexual development is regulated by chorionic gonadotropin (CG) and luteinizing hormone (LH), the action of both mediated by the LH receptor (LHR). Mutations that inactivate the LHR cause Leydig cell hypoplasia (LCH), an autosomal recessive disorder. In its mild form, LCH patients present with male hypogonadism. In its severe form, patients present with male pseudohermaphroditism, with female external genitalia, and cryptorchid testis. Mullerian derivatives are absent. Histological examination of the testis shows absence of mature Leydig cells. LCH patients have elevated plasma levels of LH, normal-to-elevated levels of follicle stimulating hormone (FSH), and low levels of testosterone that do not respond to CG stimulation. Missense mutations, nonsense mutations, deletion mutations, and in-frame insertion mutation of the LHR have been identified in patients with LCH. These mutations are not localized in any particular region of the gene and cause variable degrees of receptor-activity loss. The clinical manifestation of patients with LCH with homozygous or compound heterozygous mutations can be correlated with the residual activity of their respective mutated LHRs. Homozygous inactivating mutations of the LHR in the female cause hypergonadotrophic hypogonadism with primary amenorrhea or oligoamenorrhea, cystic ovaries, and infertility. © 2000 IMSS. Published by Elsevier Science Inc. Key Words: Luteinizing hormone receptor, Inactivating mutation, Single base substitution, Deletion, Insertion, Hypogonadism, Pseudohermaphroditism.
Introduction Human male sexual development is regulated by chorionic gonadotropin (CG) during early gestation, and by luteinizing hormone (LH) in late gestation and after puberty. CG induces differentiation of Leydig cells, while both CG and LH stimulate Leydig cells to produce testosterone. Under normal physiological conditions, testosterone is almost exclusively (.95%) of testicular origin. Testosterone plays a critical role in human male sexual development. It is obligatory for the development of male internal and external genitalia, and for the establishment and maintenance of secondary male sexual characteristics (1,2). CG and LH share a common cell surface receptor, the CG/LH receptor (LHR). The LHR is a G protein-coupled receptor. Binding of the hormone to the receptor triggers signal transduction, resulting in the activation of the adenylyl cyclase cascade and subsequent testosterone production. ReAddress reprint requests to: Wai-Yee Chan, Department of Pediatrics, Georgetown University Children’s Medical Center, 3800 Reservoir Road, NW, Washington, D.C. 20007. Tel.: (1202) 687-7068; FAX: (1202) 6871629; E-mail: [email protected]
sistance of Leydig cells to the action of CG and LH, due to inactivating mutations of the LHR, causes hypoplasia of the Leydig cells and decreased production of testosterone, giving rise to hypogonadism or male pseudohermaphroditism, called Leydig cell hypoplasia (LCH) or Leydig cell agenesis.
Clinical Features of LCH Patients LCH as a cause of male pseudohermaphroditism was first described in 1976 by Berthezene et al. (3). Since then, a number of patients with LCH have been reported. These patients usually sought medical attention, either in the prepubertal period, because testicles were palpated in the labia majora, or late in puberty, because of lack of female secondary sexual characteristics development, such as breast development and amenorrhea. A number of features distinguish LCH from the other forms of male pseudohermaphroditism. LCH is an autosomal recessive disorder. Patients are genetic males, with a 46,XY karyotype (4). Clinical presentation of LCH is variable. At one end of the spectrum are patients with normal-appearing female
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external genitalia, often leading to female sex assignment. At the other end of the spectrum are patients with hypergonadotropic hypogonadism, with microphallus and hypoplastic male external genitalia. In between are patients with variable degrees of masculinization of the external genitalia, including slight enlargement of the clitoris and its hood, along with some posterior fusion of the labia associated with the presence of the vas deferens and epidydimis (3,5–7). Based on the phenotypic differences, two forms of LCH were proposed: type 1, the severe form with male pseudohermaphroditism, and type 2, the mild form with male primary hypogonadism (7). It becomes clear that the variable manifestation of the disorder is correlated with the residual activity of the mutated LHR. In spite of phenotypic female external genitalia in the patient, testis are often detected in the labia majora or intraabdominal. The vagina is quite short and there are no uterus and fallopian tubes. Instead, epidydimis and vas deferens are present. Both descended and cryptorchid testes with relatively well-preserved seminiferous tubules and absence of mature Leydig cells have been observed in patients with LCH (3,5,6,8–14). LCH patients show no development of either male or female secondary sexual characteristics at puberty. In a patient with the mild form of LCH, exogenous testosterone therapy resulted in normal virilization (9). Hormonal Profile of Patients with LCH LCH patients have elevated serum levels of LH, normal-toelevated levels of follicle stimulating hormone (FSH), and low levels of testosterone, which is unresponsive to human CG stimulation. Primary absence of testosterone biosynthetic enzymes in these patients, such as 17-ketosteroid reductase, 17,20-desmolase, 3-b-hydroxysteroid dehydrogenase, and 17a-hydroxylase were ruled out by the demonstration of normal levels of metabolites of the testosterone biosynthetic pathway (3,5,6,8–10,12,13). Normal 5-a-reductase activity, and biologically active LH and androgen receptor were also shown in patients with LCH (5,6,8,9,11,12). Observation of decreased CG binding of gonadal tissues from the patients suggested the primary defect in LCH to be the LHR (6,8,9,12,14). Mutations of the LHR in LCH Since the first description of the inactivating mutation of the LH receptor in an LCH patient in 1994 (15), 12 different mutations of the LHR have been described to inactivate the receptor and give rise to LCH. Inactivating mutations include single-base substitutions, deletions, and in-frame insertion. The amino-acid residues affected by the inactivating mutations are shown in Figure 1. Single-Base Substitution Seven single-base substitutions leading to missense mutations have been identified in seven LCH kindreds. Among these,
three are homozygous mutations, namely, Cys131Arg (nucleotide 391-T to C) (16), Glu354Lys (nucleotide 1060-G to A) (17), and Ala593Pro (nucleotide 1777-G to C) (18), all products of consanguineous marriages. Others comprise one homozygous mutation, Ile625Lys (nucleotide 1874-T to A) in one kindred (19), and two compound heterozygous mutations, Cys343Ser (nucleotide 1027-T to A) and Cys543Arg (nucleotide 1627-T to C), in the other (20). One mutation, Ser616Tyr (nucleotide 1847-C to A) was found as a homozygous mutation in one kindred (21), and as a heterozygous mutation in another (22). Single-base substitutions that lead to nonsense mutations have been found in four kindreds. Two of them have the homozygous Arg554Stop (nucleotide 1660-C to T) mutation (21,23). One kindred has Arg554Stop as a heterozygous mutation (23), and the other has another heterozygous nonsense mutation Cys545Stop (nucleotide 1635-C to A) (24). All single-base substitutions affect amino acids, which are conserved among the LHR of human, rat, and porcine (25), and, with the exception of Cys-131, are conserved among the following three glycohormone receptors: thyroid stimulating hormone receptor (TSHR); FSH receptor (FSHR), and LHR (26). Cys-131, though not conserved, may play a role in the hormone binding or surface expression of the LHR, because extracellular cysteine residues in the LHR have been suggested to have this function (27). The effect of most of these inactivating mutations on the signal transduction activity of the LHR has been demonstrated by in vitro expression studies in either HEK 293 or COS-7 cells. Response of cells expressing mutant LHR cDNAs to hCG stimulation, in terms of cAMP synthesis, varies from mildly reduced (Ile625Lys) (19) to no synthesis [Glu354Lys (17), Cys545Stop (24), and Ala593Pro (18)]. Even though reduced cell-surface expression found in almost all cases, unaltered hormone-binding affinity was observed for several mutated LHRs, including the ones with the Ala593Pro (18), Ser616Tyr (22), and Ile625Lys mutations (19).
Deletions In compound heterozygous LCH kindred with the Ser616Tyr mutation, exon 8 of the other LHR allele was deleted (22). Exon 8 of the human LHR encodes a polypeptide of 25 amino acids, which comprise part of the VII and most of the VIII leucine-rich repeat in the extracellular domain (28). Leucine-rich repeats have been shown to form a specific, high-affinity, hormone-binding site of the receptor (29). In vitro expression studies demonstrated failure of hormone binding of the mutated receptor and drastically reduced cAMP synthesis, in response to hCG stimulation of the transfected cells (22). This confirms that deletion of exon 8 contributes to LCH in this kindred. Smaller deletion can be equally detrimental. A homozygous 6-bp deletion has been shown to be responsible for inactivating the LHR, giving rise to LCH in another kindred. A 46,XY
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Figure 1. Amino acid residues affected by inactivating mutations identified in the LHR of patients with LCH. Amino-acid residues are represented by squares or circles, with the single alphabet code of amino acids inside. Squared amino acids constitute the putative signal peptide (28). Inverted triangle indicates insertion. Filled circle represents deleted amino acids. Amino acids affected by missense mutations and nonsense mutations are shaded gray. Positions of the amino acids are indicated by the numbers next to them. From left to right, cylinders represent TM I–VII.
pseudohermaphrodite and his 46,XX sister inherited a homozygous deletion of nucleotides 1822–1827 from their consanguineous parent (30). This causes the deletion of Leu-608 and Val-609 within transmembrane helix (TM) VII of the LHR. Both Leu608 and Val-609 are highly conserved amino acids in the LHR
of different species (25) and among the human glycohormone receptors (26). In vitro expression studies showed unaltered hormone-binding affinity, but drastically reduced cell-surface expression of the mutated receptor and markedly reduced hormone-induced cAMP biosynthesis of the transfected cell (30).
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In-Frame Insertion A 33-bp in-frame insertion has been found in the maternal LHR allele in a kindred with two 46,XY male pseudohermaphrodites (31). The paternal LHR allele of the patients carries the Cys545Stop nonsense mutation (24). The insertion occurs between nucleotides 54 and 55. This region of the LHR gene is rich in the CTG trinucleotide and is unstable. The in-frame insertion is likely the result of duplication of nucleotides 28–54 immediately upstream of the insertion. At the same site, a smaller but polymorphic 6-bp insertion is present in the general population (32). The insertion occurs between amino-acid residues 18 and 19, immediately upstream of the signal peptide cleavage site (28). In vitro expression studies showed no hormone binding on the cell surface or in the cytoplasm of cells expressing the mutated LHR, suggesting that the processing and/or stability of the mutated LHR transcript is affected by the insertion (31).
Structure-Function Effect and Frequency of the Inactivating Mutations Unlike the activating mutations (33), the inactivating mutations are not localized in any particular region of the receptor and are scattered throughout the LHR gene in the extracellular domain, the third cytoplasmic loop, or TM V, VI, and VII (Figure 1). It is now known that the extracellular domain of the LHR is important for high-affinity binding of the hormone, while the transmembrane domain is important for signal transduction (27). Mutations in the extracellular domain that affect hormone binding often lead to the diminution, but not the absence, of signal transduction (16,17,22). This is probably due to the presence of low-affinity binding sites for the hormone in the transmembrane domain of the receptor (27). Thus, mutations in the extracellular domain, such as Cys131Arg (16), Glu354Lys (17), and exon 8 deletion (22) have led to impaired cAMP response of cells expressing LHR with such mutations. On the other hand, the effect of mutations in the transmembrane domain on the signal transduction activity of the receptor is more variable, depending on the role
of the affected amino-acid residue in processes such as trafficking and coupling efficiency (27,34–36). For example, cell-surface expression of the LHR appears to be affected by the presence of the Ser616Tyr mutation (22), and coupling efficiency appears to be affected by mutations such as Ala593Pro (18) and Ile625Lys (19). Deletion of a couple of amino acids in this region (30), or nonsense mutations that cause premature truncation of the receptor, (21,23,24) are likely to be more disruptive, causing abolition of signal transduction. For example, Cys545Stop mutation (24) causes truncation of the receptor and the loss of TM VI (known to be critical for signal transduction (34)) and residues in the cytoplasmic C-domain (shown to be critical for cell surface expression of the receptor (35,36)). Cells expressing LHR with the Cys545Stop mutation did not respond to hCG stimulation (24). Due to the small number of LCH chromosomes analyzed, the frequency of the different mutations in LCH cannot be calculated. There seems to be no predominant form of inactivating mutation. Nonetheless, it is noteworthy that Arg554Stop mutation was found in three unrelated families and Ser616Tyr mutation was found in two (21,22). Genotype-Phenotype Correlation in LCH In LCH, there is apparent correlation between the severity of the clinical phenotype and the amount of residual activity of the mutated LHR. The relationship between clinical phenotype and residual activity of the mutated LHR in the patient with LCH is compared in Table 1. The severe phenotype, i.e., male pseudohermaphroditism or type 1 LCH (7), is manifested in patients with mutated LHRs that either fail to be expressed on the cell surface or are unable to transduce the signal of hormone binding. Thus, patients who have homozygous Glu354Lys (17), Ala593Pro (18), and Leu-608-Val-609 deletion (30) and compound heterozygous Cys545Stop/insertion mutations (24,31), are male pseudohermaphrodites with female external genitalia and undescended testis. Milder forms of LCH, i.e., those with male hypogonadism or type 2 LCH (7), are manifested in patients with mutated LHRs that have reduced, but not eliminated, cell-surface hCG binding and signal transduction,
Table 1. Genotype-phenotype correlation in Leydig cell hypoplasia Genotype S616Y/S616Y I625K/I625K S616Y/nexon 8 C131R/C131R n608L-609V/n608L-609V E354K/E354K A593P/A593P C545*/541Ins
hCG affinity
cAMP response
Phenotype
Reference
Normal Normal Normal/reduced Reduced Normal Normal Normal Reduced
Reduced Reduced Reduced Reduced Greatly reduced No No No
Male, hypogonadism Male, hypogonadism Male, hypogonadism, severe hypospadias Sexual ambiguity at birth Female, pseudohermaphroditism Female, pseudohermaphroditism Female, pseudohermaphroditism Female, pseudohermaphroditism
(21) (19) (22) (16) (30) (17) (18) (24, 31)
Genotype of each LHR allele is separated by (/); one-letter symbols are used for amino acids; (*) is used to indicate a STOP codon; (n) indicates deletion; “54 1 Ins” indicates insertion after nucleotide 54.
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such as in Cys131Arg (16), Ser616Tyr (22), Ile625Lys (21), and exon 8 deletion (22). Therefore, patients with homozygous Cys131Arg mutation, homozygous Ile625Lys mutation, or compound heterozygous Ser616Tyr/exon 8 deletion mutations have a micropenis and hypospadias (16,19,22). Another patient with homozygous Ser616Tyr mutation also exhibits micropenis, hypospadias, and descended testes (21). However, in the latter case, investigators reported absence of ligand binding when the mutated LHR was expressed in COS-7 cells. It is not yet clear why the results of in vitro expression studies of the Ser616Tyr differ in this case from previous studies (22).
Effect of Inactivation of the LHR in Females Human female sexual differentiation and pubertal development do not depend on the action of LH. LH is sufficient enough to trigger steroidogenesis in Leydig cells, but both LH and FSH are required to activate ovarian steroidogenesis. LH promotes follicular maturation and ovulation, and during the luteal phase induces the formation of the corpus luteum and stimulates progesterone secretion (37). Inactivation of the LHR, resulting in abnormal LH action, will cause defective follicular development, ovulation, amenorrhea, and infertility. Due to the later onset time and milder disease phenotype, 46,XX females with homozygous or compound heterozygous inactivating mutations of the LHR are more difficult to identify unless an LCH male sibling is present. Identification of 46,XX females with inactivating LHR mutations led Toledo et al. to propose a third phenotype, type 3, of LCH (38). The first 46,XX female affected by inactivating mutations of the LHR was identified as the sister of three LCH 46,XY male pseudohermaphrodites with homozygous Arg554Stop mutation. The patient presented with normal breast and pubic hair development, a small uterus, cystic ovary, and secondary amenorrhea. She had elevated plasma levels of LH and normal levels of FSH, but low levels of estradiol and progesterone (21). The 46,XX female in another LCH kindred with homozygous Ala593Pro mutation of the LHR also showed normal development of female internal and external genitalia, primary amenorrhea, and infertility. In addition to an elevated plasma FSH level, the hormonal profile was the same as the first patient. Histological examination of ovarian tissues revealed complete follicular development, but no signs of ovulation (39). The third 46,XX female with homozygous Leu-608-Val-609 deletion inactivating mutation of the LHR showed menstrual irregularities (oligoamenorrhea), enlarged cystic ovary, and infertility (30). A fourth 46,XX female with homozygous Glu354Lys inactivating mutation had normal female phenotype, good breast development, cystic ovary, and primary amenorrhea (17). Thus, 46,XX females with homozygous or compound heterozygous inactivating mutations of the LHR all show normal breast and pubic hair development at puberty, develop hypergonadotrophic hypogonadism, have amenorrhea
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or oligoamenorrhea and enlarged cystic ovaries, and are infertile. Although they manifest ovarian resistance to LH, follicular development is not compromised (23). All these observations are consistent with the independence of female sexual differentiation and pubertal development on LH, the primary roles of FSH on folliculogenesis and LH on ovulation, and the formation of the corpus luteum. Conclusions LCH is an autosomal recessive disorder in which loss of function of the LHR in the male prevents normal sexual development, giving rise to hypergonadotrophic hypogonadism or male pseudohermaphroditism. Single-base substitutions, deletions, and insertions have been identified in the LHR gene in LCH patients. These mutations reduce or abolish the signal transduction activity of the LHR, causing testicular and ovarian resistance to LH. The variable degree of manifestation of the condition depends on the effect of the mutation on the biological activity of the LHR. Mutations that inactivate the receptor cause pseudohermaphroditism with normal-appearing female external genitalia, while mutations that reduce the activity of the receptor cause hypogandism with micropenis. Milder phenotypes are observed in females with homozygous or compound heterozygous inactivating mutations of the LHR. This difference in the effect of LHR mutations on the male and female confirms the different roles of the LH in the sexual development of the two sexes. Studies of the effect of different mutations on the biological activity of the LHR will lead to better understanding of the structure-function relationship of the LHR. Knowledge of the genotype-phenotype correlation in LCH will help diagnosis, prognosis, and the design of better treatment protocol for patients with this disorder. Acknowledgments We are indebted to Dr. Louisa Laue, for introducing us to the interesting field of LH receptor research; to Dr. Owen M. Rennert, for his support throughout the years; to Dr. Aaron JW Hsueh, for his generous help and advice; to Dr. Gordon B. Cutler, Jr., for his helpful discussions; and to all those collaborators who generously referred their patients to us for our study.
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REVIEW ARTICLE
Male Pseudohermaphroditism Due to Inactivating Luteinizing Hormone Receptor Mutations Shao-Ming Wu* and Wai-Yee Chan**,*** Departments of *Pediatrics, **Biochemistry & Molecular Biology, and ***Cell Biology, Georgetown University, Washington, DC, USA Received for publication September 9, 1999; accepted September 9, 1999 (99/159).
Human male sexual development is regulated by chorionic gonadotropin (CG) and luteinizing hormone (LH), the action of both mediated by the LH receptor (LHR). Mutations that inactivate the LHR cause Leydig cell hypoplasia (LCH), an autosomal recessive disorder. In its mild form, LCH patients present with male hypogonadism. In its severe form, patients present with male pseudohermaphroditism, with female external genitalia, and cryptorchid testis. Mullerian derivatives are absent. Histological examination of the testis shows absence of mature Leydig cells. LCH patients have elevated plasma levels of LH, normal-to-elevated levels of follicle stimulating hormone (FSH), and low levels of testosterone that do not respond to CG stimulation. Missense mutations, nonsense mutations, deletion mutations, and in-frame insertion mutation of the LHR have been identified in patients with LCH. These mutations are not localized in any particular region of the gene and cause variable degrees of receptor-activity loss. The clinical manifestation of patients with LCH with homozygous or compound heterozygous mutations can be correlated with the residual activity of their respective mutated LHRs. Homozygous inactivating mutations of the LHR in the female cause hypergonadotrophic hypogonadism with primary amenorrhea or oligoamenorrhea, cystic ovaries, and infertility. © 2000 IMSS. Published by Elsevier Science Inc. Key Words: Luteinizing hormone receptor, Inactivating mutation, Single base substitution, Deletion, Insertion, Hypogonadism, Pseudohermaphroditism.
Introduction Human male sexual development is regulated by chorionic gonadotropin (CG) during early gestation, and by luteinizing hormone (LH) in late gestation and after puberty. CG induces differentiation of Leydig cells, while both CG and LH stimulate Leydig cells to produce testosterone. Under normal physiological conditions, testosterone is almost exclusively (.95%) of testicular origin. Testosterone plays a critical role in human male sexual development. It is obligatory for the development of male internal and external genitalia, and for the establishment and maintenance of secondary male sexual characteristics (1,2). CG and LH share a common cell surface receptor, the CG/LH receptor (LHR). The LHR is a G protein-coupled receptor. Binding of the hormone to the receptor triggers signal transduction, resulting in the activation of the adenylyl cyclase cascade and subsequent testosterone production. ReAddress reprint requests to: Wai-Yee Chan, Department of Pediatrics, Georgetown University Children’s Medical Center, 3800 Reservoir Road, NW, Washington, D.C. 20007. Tel.: (1202) 687-7068; FAX: (1202) 6871629; E-mail: [email protected]
sistance of Leydig cells to the action of CG and LH, due to inactivating mutations of the LHR, causes hypoplasia of the Leydig cells and decreased production of testosterone, giving rise to hypogonadism or male pseudohermaphroditism, called Leydig cell hypoplasia (LCH) or Leydig cell agenesis.
Clinical Features of LCH Patients LCH as a cause of male pseudohermaphroditism was first described in 1976 by Berthezene et al. (3). Since then, a number of patients with LCH have been reported. These patients usually sought medical attention, either in the prepubertal period, because testicles were palpated in the labia majora, or late in puberty, because of lack of female secondary sexual characteristics development, such as breast development and amenorrhea. A number of features distinguish LCH from the other forms of male pseudohermaphroditism. LCH is an autosomal recessive disorder. Patients are genetic males, with a 46,XY karyotype (4). Clinical presentation of LCH is variable. At one end of the spectrum are patients with normal-appearing female
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external genitalia, often leading to female sex assignment. At the other end of the spectrum are patients with hypergonadotropic hypogonadism, with microphallus and hypoplastic male external genitalia. In between are patients with variable degrees of masculinization of the external genitalia, including slight enlargement of the clitoris and its hood, along with some posterior fusion of the labia associated with the presence of the vas deferens and epidydimis (3,5–7). Based on the phenotypic differences, two forms of LCH were proposed: type 1, the severe form with male pseudohermaphroditism, and type 2, the mild form with male primary hypogonadism (7). It becomes clear that the variable manifestation of the disorder is correlated with the residual activity of the mutated LHR. In spite of phenotypic female external genitalia in the patient, testis are often detected in the labia majora or intraabdominal. The vagina is quite short and there are no uterus and fallopian tubes. Instead, epidydimis and vas deferens are present. Both descended and cryptorchid testes with relatively well-preserved seminiferous tubules and absence of mature Leydig cells have been observed in patients with LCH (3,5,6,8–14). LCH patients show no development of either male or female secondary sexual characteristics at puberty. In a patient with the mild form of LCH, exogenous testosterone therapy resulted in normal virilization (9). Hormonal Profile of Patients with LCH LCH patients have elevated serum levels of LH, normal-toelevated levels of follicle stimulating hormone (FSH), and low levels of testosterone, which is unresponsive to human CG stimulation. Primary absence of testosterone biosynthetic enzymes in these patients, such as 17-ketosteroid reductase, 17,20-desmolase, 3-b-hydroxysteroid dehydrogenase, and 17a-hydroxylase were ruled out by the demonstration of normal levels of metabolites of the testosterone biosynthetic pathway (3,5,6,8–10,12,13). Normal 5-a-reductase activity, and biologically active LH and androgen receptor were also shown in patients with LCH (5,6,8,9,11,12). Observation of decreased CG binding of gonadal tissues from the patients suggested the primary defect in LCH to be the LHR (6,8,9,12,14). Mutations of the LHR in LCH Since the first description of the inactivating mutation of the LH receptor in an LCH patient in 1994 (15), 12 different mutations of the LHR have been described to inactivate the receptor and give rise to LCH. Inactivating mutations include single-base substitutions, deletions, and in-frame insertion. The amino-acid residues affected by the inactivating mutations are shown in Figure 1. Single-Base Substitution Seven single-base substitutions leading to missense mutations have been identified in seven LCH kindreds. Among these,
three are homozygous mutations, namely, Cys131Arg (nucleotide 391-T to C) (16), Glu354Lys (nucleotide 1060-G to A) (17), and Ala593Pro (nucleotide 1777-G to C) (18), all products of consanguineous marriages. Others comprise one homozygous mutation, Ile625Lys (nucleotide 1874-T to A) in one kindred (19), and two compound heterozygous mutations, Cys343Ser (nucleotide 1027-T to A) and Cys543Arg (nucleotide 1627-T to C), in the other (20). One mutation, Ser616Tyr (nucleotide 1847-C to A) was found as a homozygous mutation in one kindred (21), and as a heterozygous mutation in another (22). Single-base substitutions that lead to nonsense mutations have been found in four kindreds. Two of them have the homozygous Arg554Stop (nucleotide 1660-C to T) mutation (21,23). One kindred has Arg554Stop as a heterozygous mutation (23), and the other has another heterozygous nonsense mutation Cys545Stop (nucleotide 1635-C to A) (24). All single-base substitutions affect amino acids, which are conserved among the LHR of human, rat, and porcine (25), and, with the exception of Cys-131, are conserved among the following three glycohormone receptors: thyroid stimulating hormone receptor (TSHR); FSH receptor (FSHR), and LHR (26). Cys-131, though not conserved, may play a role in the hormone binding or surface expression of the LHR, because extracellular cysteine residues in the LHR have been suggested to have this function (27). The effect of most of these inactivating mutations on the signal transduction activity of the LHR has been demonstrated by in vitro expression studies in either HEK 293 or COS-7 cells. Response of cells expressing mutant LHR cDNAs to hCG stimulation, in terms of cAMP synthesis, varies from mildly reduced (Ile625Lys) (19) to no synthesis [Glu354Lys (17), Cys545Stop (24), and Ala593Pro (18)]. Even though reduced cell-surface expression found in almost all cases, unaltered hormone-binding affinity was observed for several mutated LHRs, including the ones with the Ala593Pro (18), Ser616Tyr (22), and Ile625Lys mutations (19).
Deletions In compound heterozygous LCH kindred with the Ser616Tyr mutation, exon 8 of the other LHR allele was deleted (22). Exon 8 of the human LHR encodes a polypeptide of 25 amino acids, which comprise part of the VII and most of the VIII leucine-rich repeat in the extracellular domain (28). Leucine-rich repeats have been shown to form a specific, high-affinity, hormone-binding site of the receptor (29). In vitro expression studies demonstrated failure of hormone binding of the mutated receptor and drastically reduced cAMP synthesis, in response to hCG stimulation of the transfected cells (22). This confirms that deletion of exon 8 contributes to LCH in this kindred. Smaller deletion can be equally detrimental. A homozygous 6-bp deletion has been shown to be responsible for inactivating the LHR, giving rise to LCH in another kindred. A 46,XY
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Figure 1. Amino acid residues affected by inactivating mutations identified in the LHR of patients with LCH. Amino-acid residues are represented by squares or circles, with the single alphabet code of amino acids inside. Squared amino acids constitute the putative signal peptide (28). Inverted triangle indicates insertion. Filled circle represents deleted amino acids. Amino acids affected by missense mutations and nonsense mutations are shaded gray. Positions of the amino acids are indicated by the numbers next to them. From left to right, cylinders represent TM I–VII.
pseudohermaphrodite and his 46,XX sister inherited a homozygous deletion of nucleotides 1822–1827 from their consanguineous parent (30). This causes the deletion of Leu-608 and Val-609 within transmembrane helix (TM) VII of the LHR. Both Leu608 and Val-609 are highly conserved amino acids in the LHR
of different species (25) and among the human glycohormone receptors (26). In vitro expression studies showed unaltered hormone-binding affinity, but drastically reduced cell-surface expression of the mutated receptor and markedly reduced hormone-induced cAMP biosynthesis of the transfected cell (30).
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In-Frame Insertion A 33-bp in-frame insertion has been found in the maternal LHR allele in a kindred with two 46,XY male pseudohermaphrodites (31). The paternal LHR allele of the patients carries the Cys545Stop nonsense mutation (24). The insertion occurs between nucleotides 54 and 55. This region of the LHR gene is rich in the CTG trinucleotide and is unstable. The in-frame insertion is likely the result of duplication of nucleotides 28–54 immediately upstream of the insertion. At the same site, a smaller but polymorphic 6-bp insertion is present in the general population (32). The insertion occurs between amino-acid residues 18 and 19, immediately upstream of the signal peptide cleavage site (28). In vitro expression studies showed no hormone binding on the cell surface or in the cytoplasm of cells expressing the mutated LHR, suggesting that the processing and/or stability of the mutated LHR transcript is affected by the insertion (31).
Structure-Function Effect and Frequency of the Inactivating Mutations Unlike the activating mutations (33), the inactivating mutations are not localized in any particular region of the receptor and are scattered throughout the LHR gene in the extracellular domain, the third cytoplasmic loop, or TM V, VI, and VII (Figure 1). It is now known that the extracellular domain of the LHR is important for high-affinity binding of the hormone, while the transmembrane domain is important for signal transduction (27). Mutations in the extracellular domain that affect hormone binding often lead to the diminution, but not the absence, of signal transduction (16,17,22). This is probably due to the presence of low-affinity binding sites for the hormone in the transmembrane domain of the receptor (27). Thus, mutations in the extracellular domain, such as Cys131Arg (16), Glu354Lys (17), and exon 8 deletion (22) have led to impaired cAMP response of cells expressing LHR with such mutations. On the other hand, the effect of mutations in the transmembrane domain on the signal transduction activity of the receptor is more variable, depending on the role
of the affected amino-acid residue in processes such as trafficking and coupling efficiency (27,34–36). For example, cell-surface expression of the LHR appears to be affected by the presence of the Ser616Tyr mutation (22), and coupling efficiency appears to be affected by mutations such as Ala593Pro (18) and Ile625Lys (19). Deletion of a couple of amino acids in this region (30), or nonsense mutations that cause premature truncation of the receptor, (21,23,24) are likely to be more disruptive, causing abolition of signal transduction. For example, Cys545Stop mutation (24) causes truncation of the receptor and the loss of TM VI (known to be critical for signal transduction (34)) and residues in the cytoplasmic C-domain (shown to be critical for cell surface expression of the receptor (35,36)). Cells expressing LHR with the Cys545Stop mutation did not respond to hCG stimulation (24). Due to the small number of LCH chromosomes analyzed, the frequency of the different mutations in LCH cannot be calculated. There seems to be no predominant form of inactivating mutation. Nonetheless, it is noteworthy that Arg554Stop mutation was found in three unrelated families and Ser616Tyr mutation was found in two (21,22). Genotype-Phenotype Correlation in LCH In LCH, there is apparent correlation between the severity of the clinical phenotype and the amount of residual activity of the mutated LHR. The relationship between clinical phenotype and residual activity of the mutated LHR in the patient with LCH is compared in Table 1. The severe phenotype, i.e., male pseudohermaphroditism or type 1 LCH (7), is manifested in patients with mutated LHRs that either fail to be expressed on the cell surface or are unable to transduce the signal of hormone binding. Thus, patients who have homozygous Glu354Lys (17), Ala593Pro (18), and Leu-608-Val-609 deletion (30) and compound heterozygous Cys545Stop/insertion mutations (24,31), are male pseudohermaphrodites with female external genitalia and undescended testis. Milder forms of LCH, i.e., those with male hypogonadism or type 2 LCH (7), are manifested in patients with mutated LHRs that have reduced, but not eliminated, cell-surface hCG binding and signal transduction,
Table 1. Genotype-phenotype correlation in Leydig cell hypoplasia Genotype S616Y/S616Y I625K/I625K S616Y/nexon 8 C131R/C131R n608L-609V/n608L-609V E354K/E354K A593P/A593P C545*/541Ins
hCG affinity
cAMP response
Phenotype
Reference
Normal Normal Normal/reduced Reduced Normal Normal Normal Reduced
Reduced Reduced Reduced Reduced Greatly reduced No No No
Male, hypogonadism Male, hypogonadism Male, hypogonadism, severe hypospadias Sexual ambiguity at birth Female, pseudohermaphroditism Female, pseudohermaphroditism Female, pseudohermaphroditism Female, pseudohermaphroditism
(21) (19) (22) (16) (30) (17) (18) (24, 31)
Genotype of each LHR allele is separated by (/); one-letter symbols are used for amino acids; (*) is used to indicate a STOP codon; (n) indicates deletion; “54 1 Ins” indicates insertion after nucleotide 54.
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such as in Cys131Arg (16), Ser616Tyr (22), Ile625Lys (21), and exon 8 deletion (22). Therefore, patients with homozygous Cys131Arg mutation, homozygous Ile625Lys mutation, or compound heterozygous Ser616Tyr/exon 8 deletion mutations have a micropenis and hypospadias (16,19,22). Another patient with homozygous Ser616Tyr mutation also exhibits micropenis, hypospadias, and descended testes (21). However, in the latter case, investigators reported absence of ligand binding when the mutated LHR was expressed in COS-7 cells. It is not yet clear why the results of in vitro expression studies of the Ser616Tyr differ in this case from previous studies (22).
Effect of Inactivation of the LHR in Females Human female sexual differentiation and pubertal development do not depend on the action of LH. LH is sufficient enough to trigger steroidogenesis in Leydig cells, but both LH and FSH are required to activate ovarian steroidogenesis. LH promotes follicular maturation and ovulation, and during the luteal phase induces the formation of the corpus luteum and stimulates progesterone secretion (37). Inactivation of the LHR, resulting in abnormal LH action, will cause defective follicular development, ovulation, amenorrhea, and infertility. Due to the later onset time and milder disease phenotype, 46,XX females with homozygous or compound heterozygous inactivating mutations of the LHR are more difficult to identify unless an LCH male sibling is present. Identification of 46,XX females with inactivating LHR mutations led Toledo et al. to propose a third phenotype, type 3, of LCH (38). The first 46,XX female affected by inactivating mutations of the LHR was identified as the sister of three LCH 46,XY male pseudohermaphrodites with homozygous Arg554Stop mutation. The patient presented with normal breast and pubic hair development, a small uterus, cystic ovary, and secondary amenorrhea. She had elevated plasma levels of LH and normal levels of FSH, but low levels of estradiol and progesterone (21). The 46,XX female in another LCH kindred with homozygous Ala593Pro mutation of the LHR also showed normal development of female internal and external genitalia, primary amenorrhea, and infertility. In addition to an elevated plasma FSH level, the hormonal profile was the same as the first patient. Histological examination of ovarian tissues revealed complete follicular development, but no signs of ovulation (39). The third 46,XX female with homozygous Leu-608-Val-609 deletion inactivating mutation of the LHR showed menstrual irregularities (oligoamenorrhea), enlarged cystic ovary, and infertility (30). A fourth 46,XX female with homozygous Glu354Lys inactivating mutation had normal female phenotype, good breast development, cystic ovary, and primary amenorrhea (17). Thus, 46,XX females with homozygous or compound heterozygous inactivating mutations of the LHR all show normal breast and pubic hair development at puberty, develop hypergonadotrophic hypogonadism, have amenorrhea
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or oligoamenorrhea and enlarged cystic ovaries, and are infertile. Although they manifest ovarian resistance to LH, follicular development is not compromised (23). All these observations are consistent with the independence of female sexual differentiation and pubertal development on LH, the primary roles of FSH on folliculogenesis and LH on ovulation, and the formation of the corpus luteum. Conclusions LCH is an autosomal recessive disorder in which loss of function of the LHR in the male prevents normal sexual development, giving rise to hypergonadotrophic hypogonadism or male pseudohermaphroditism. Single-base substitutions, deletions, and insertions have been identified in the LHR gene in LCH patients. These mutations reduce or abolish the signal transduction activity of the LHR, causing testicular and ovarian resistance to LH. The variable degree of manifestation of the condition depends on the effect of the mutation on the biological activity of the LHR. Mutations that inactivate the receptor cause pseudohermaphroditism with normal-appearing female external genitalia, while mutations that reduce the activity of the receptor cause hypogandism with micropenis. Milder phenotypes are observed in females with homozygous or compound heterozygous inactivating mutations of the LHR. This difference in the effect of LHR mutations on the male and female confirms the different roles of the LH in the sexual development of the two sexes. Studies of the effect of different mutations on the biological activity of the LHR will lead to better understanding of the structure-function relationship of the LHR. Knowledge of the genotype-phenotype correlation in LCH will help diagnosis, prognosis, and the design of better treatment protocol for patients with this disorder. Acknowledgments We are indebted to Dr. Louisa Laue, for introducing us to the interesting field of LH receptor research; to Dr. Owen M. Rennert, for his support throughout the years; to Dr. Aaron JW Hsueh, for his generous help and advice; to Dr. Gordon B. Cutler, Jr., for his helpful discussions; and to all those collaborators who generously referred their patients to us for our study.
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