Lossof Function Mutations of GPR54

Congenital isolated hypogonadotropic hypogonadism (IHH) is defined as a deficiency of the pituitary secretion of LH and FSH, which results from defects in the synthesis, secretion, and action of GnRH, leading to impairment of pubertal maturation and reproductive function.71 It is characterized by low levels of sex steroids in the presence of low or inappropriately normal LH and

FSH serum levels, with no anatomical lesion in the hypothalamo-pituitary tract and no other associated pituitary hormone deficiency.72 It is called Kallmann syndrome when associated to anosmia.73 IHH is an infrequent and heterogeneous disorder, which can be sporadic or inherited as, autosomal recessive, dominant, or an X-linked trait. IHH is usually diagnosed in the second or third decade of life, presenting with delayed pubertal development, primary amen-orrhea, or infertility.72 The presence of microphallus and cryptorchidism at birth or in younger age may disclose the diagnosis before puberty.73

An increasing number of genes has been described to be involved in the pathogenesis of IHH with normal sense of smell, including defects in GnRH receptor (GnRHR), in fibroblast growth factor receptor-1 (FGFR1), in proki-neticin (PROK2) and neurokinin B (TAC3), and their respective receptors (PROKR2 and TACR).74-77

In 2003, using the linkage analysis methodology, two different groups first described GPR54 loss-of-function mutations in patients with normosmic IHH.7,8 de Roux et al.8 described one consanguineous family with five affected members that harbored a 155 bp deletion in homozygous state within the transition of intron 4 and exon 5 of the GPR54 gene. The unaffected members were heterozygous or homozygous for the wild-type sequence.8 This deletion removes the G protein binding domain which is located on the third intracel-lular loop of GPR54. Therefore, even if present on the cell surface and able to bind kisspeptin, receptors carrying this deletion are unable to couple to G proteins and thus incapable of signaling.8

Seminara et al.7 reported one novel homozygous mutation in six members with normosmic IHH of a large consanguineous Saudi Arabian family. The four males and two females with IHH harbored a missense mutation in homozygous state within exon 3 of GPR54 gene. The nucleotide thymine was changed by a cytosine resulting in the substitution of a leucine by a serine at position 148 (L148S). This amino acid change takes place in the second intracellular loop of GPR54 receptor, near the DRW/Y motif. This motif has been shown to regulate the activation and inactivation of a number of G protein-coupled receptors. The critical role of the DRW/Y motif is highlighted by several descriptions of mutations in and around this motif that result in constitutive activation or inactivation of the mutated receptors.78 In fact, functional studies indicate that inositol phosphate production by the L148S GPR54 mutant is severely impaired.7 Interestingly, the mutation of an analogous leucine on the a1-adrenergic receptor has been recently reported to equally impair receptor function, indicating that leucine at this position is required for adequate receptor activation.79 On the other hand, expression levels and trafficking of L148S GPR54 mutant to the cell surface have been reported to be similar to those of the wild-type GPR54. Using tagged receptors, Wacker et al.79 demonstrated that immunoprecipitation of L148S GPR54 mutant is comparable to that of the wild-type GPR54, indicating that this mutation does not affect receptor expression. Likewise, confocal imaging and cell sorting studies show that trafficking of the L148S GPR54 mutant to the plasma membrane and its cellular localization are not altered.79

Two other mutations (R331X and X399R) in compound heterozygous state were identified in an Afro-American patient, but in this case, with sporadic IHH in Seminara's study.7 The first mutation was an insertion of a stop codon at position 331 on the beginning of the intracellular carboxyl tail, whereas the second mutation was the substitution of the expected stop codon of GPR54 by an arginine. The signaling capacity of both mutants individually transfected into COS-7 cells as measured by kisspeptin-stimulated inositol phosphate production in transfected cells was reported to be profoundly impaired.7 The introduction of the stop codon at position 331 would, at best, produce a truncated GPR54 receptor missing the entire intracellular carboxyl tail. Such deletion is expected to severely disrupt receptor signaling. On the other hand, the replacement of the normal stop codon (X399R) is predicted to affect stability and may result in protein misfolding followed by redirection of this abnormal GPR54 to destruction. Interestingly, all the affected patients described by Seminara etal.7 had at least a partial response to acute GnRH stimulation test. In fact, the Afro-American patient with sporadic normosmic IHH due to compound heterozygous mutations exhibited a leftward-shifted dose response curve when compared to other patients with IHH without GPR54 mutations.7

Since then, four other inactivating GPR54 mutations were described in patients with normosmic IHH (Table I). Semple et al.11 described one sporadic case of normosmic IHH harboring a compound heterozygous mutation in GPR54. The boy had micropenis and undescended testes at birth and undetectable serum gonadotropins at 2 months of age. The first variant was a transition c.667T > C in exon 4, leading in a substitution of a cysteine near the cytoplasmic end of the fifth transmembrane helix for arginine (C223R), while the second mutation was a transversion in exon 5, c.891 G > T, resulting to the substitution of an arginine in the third extracellular loop for leucine (R297L). The proband's mother was heterozygous for the R297L mutation, and experienced menarche at 11 years of age. His father is believed to have normal reproductive function but was not available for testing.11 There was no family history of hypogonadism. Cysteine at the position 223 is evolutionarily conserved from fish to humans, suggesting physiological relevance. The importance of this cysteine for GPR54 function was reiterated by the lack of calcium signaling of GPR54 receptors carrying this mutation. On the other hand, the arginine on position 297 is not highly conserved; and calcium signaling by this GPR54 mutant was only mildly affected.11 Nevertheless, this mild effect was enough to result in hypogonadism when combined with the C223R amino acid GPR54 substitution.11

TABLE I

Human GPR54 Inactivating Mutations Associated with Normosmic Hypogonadotropic Hypogonadism

Case

Mutation (cDNA)

Mutation (protein)

Patient's status

Familial/sporadic

Affected members

References

1

IVS4-13- 142del155

Truncated protein

Homozygous

Familial

4M/1F*

8

2

c.443T > C

L148S

Homozygous

Familial

4M

7

3

c.991C > T/c.1195T > A

R331X/X399R

Compound heterozygous

Sporadic

M

7

4

c.667T > C/c.891G > T

C223R/R297L

Compound heterozygous

Sporadic

M

11

5

c.1101_1002insC

Frameshift

Homozygous

sporadic

M

6

6

c.305T > C

L102P

Homozygous

Familial

1M

9

7

c.305T > C

L102P

Homozygous

Familial

2M

9

8

IVS2-4-2delGC insACCGGCT

Truncated protein

Homozygous

Familial

2M

10

The first frameshift mutation in GPR54 was described by Lanfranco et al6 It was a homozygous insertion of a cytosine after nucleotide position 1001 (1001_1002insC). This insertion resulted in a frameshift in the open reading frame with elongation of the GPR54 protein from 398 to 441 amino acids.6 His parents were cousins of second degree and had German descendency. There was no report of family history of hypogonadism or infertility. He suffered from delayed puberty, bilateral undescended testes, and presented with mild hypospadia. He had low levels of LH and FSH together with low testosterone levels. His gonadotropin response to GnRH acute stimulation test was pubertal. After a 2-year GnRH pulsatile treatment he fathered a healthy male child.6

More recently, a novel GPR54 missense mutation was described by Tenenbaum-Rakover et al.9 A substitution in homozygous state of a cytosine for thymidine 305 resulted in the change of a proline by a leucine at position 102 (L102P) of the first extracellular loop. This mutation was described in two families with three and four affected members with normosmic IHH, respectively.9 Affected patients were all born from consanguineous parents. In vitro studies of the L102P mutant showed that despite displaying normal affinity for kisspeptin, maximal binding to this ligand was shown to be approximately 50% decreased when compared to the wild-type GPR54, suggesting that membrane levels of the L102P GPR54 mutant are reduced by half. In addition, kisspeptin-stimulated signaling (as measured by inositol phosphate accumulation) of the L148S GPR54 mutant was absent.9 Figure 1 shows the localization of the inactivating mutations reported in the coding region of the GPR54 receptor.

Recently, we described one novel homozygous GPR54 gene mutation in two Brazilian siblings with normosmic IHH. These two siblings carrying the new mutation in GPR54 gene were born to apparently nonconsanguineuos parents. They presented with micropenis and had no secondary sexual characteristics at the age of 14 and 18 years. Both had low testosterone levels (16.5 and 24 ng/dL) and prepubertal levels of basal gonadotropins.10 This new variant was an insertion/deletion (indel) mutation characterized by the deletion of three nucleotides (GCA) at position —2 to —4, and insertion of seven nucleotides (ACCGGCT) in the 3' splice acceptor site of intron 2 of GPR54 gene.10 The nucleotide changes were absent in a control population of 120 ethnically matched controls. Their mother carried the same mutation in the heterozygous state and reported normal pubertal development. Their father is believed to have had normal reproductive maturation and function, but was not available for testing. This complex indel mutation removed the 3' splice acceptor site of intron 2. Computational splice site prediction confirms that the substitution of the GCA nucleotides by ACCGGCT in the GPR54 mutated patients could result in aberrant intron retention or exon 3 skipping due to disruption of the constitutive site and the use of cryptic splice acceptor sites. Both events would result in truncated receptors.10 Ongoing in vitro studies will confirm this prediction.

GPR54 mutations are an infrequent cause of IHH, accounting for less than 5% of all reported normosmic IHH cases. This prevalence is significantly higher in familial cases. GPR54 alterations were identified in 3 out of 180 sporadic IHH patients (1.6%) and 5 of 24 familial cases (20.8%).

Patients with inactivating GPR54 mutations present with delayed puberty and apparently no associated condition. Cryptorchidism and micropenis are described in some cases, suggesting that the kisspetin-GPR54 system plays an important role in the testosterone production during late fetal and early neonatal sexual development.6-11 The acute response to GnRH acute stimulation test was variable among patients with GPR54 mutations and ranged from totally blunted to normal.6,7,9 A 9-year follow-up of the affected patient with the L102P mutation revealed progressive changes in pituitary response.9 The LH and FSH levels after GnRH stimulus progressively increased across the follow-up from prepubertal to almost pubertal levels around 21 years of age. This finding suggests that the L102P mutation leads to a more quantitative than qualitative defect of gonadotropic axis activation.9 Notably, treatment with chronic pulsatile GnRH therapy leaded patients to increase sperm maturation and reach fertility.6,7,9 In addition, one female patient with homozygous mutations in GPR54 had multiple conceptions, uncomplicated pregnancies and deliveries of healthy children, and lactation for several months postpartum.80

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