Abstract

The case involved a 32-year-old male patient who experienced delayed puberty until the age of 18. He exhibited low levels of LH, FSH, and testosterone. Chromosome karyotyping revealed a 46XY genotype, while whole exon sequencing identified a missense mutation (c.337T>C) in the PROKR2 gene. Pituitary MRI did not indicate the presence of any masses, leading to a clinical diagnosis of Congenital Hypogonadotropic Hypogonadism (CHH). The patient underwent hormone replacement therapy with HCG for 3 years, followed by long-term testosterone treatment. Interestingly, the patient demonstrated spontaneous sperm production without the need for gonadotropin therapy. His wife conceived naturally on four occasions, resulting in two miscarriages and two successful deliveries, indicating a reversal of the patient’s gonadal function. Bioinformatics analysis and analysis based on the guidelines provided by the American College of Medical Genetics and Genomics (ACMG) confirmed that the missense mutation c.337T>C in the PROKR2 gene was a potential pathogenic variant. However, our study indicated that this specific mutation in the PROKR2 gene could not solely account for the observed reversal of gonadal function in this patient.

Keywords: Congenital hypogonadotropic hypogonadism; Reversal; PROKR2 gene.

Citation: Ding C, Wu C, Hu D, Li F. Analysis of genotype and phenotype in congenital hypogonadotropic hypogonadism with reversible gonadotropin deficiency: A case report. J Clin Images Med Case Rep. 2023; 4(11): 2675.

Introduction

Congenital Hypogonadotropic Hypogonadism (CHH) is a rare condition characterized by insufficient production, secretion, or action of Gonadotropin-Releasing Hormone (GnRH), while the anterior pituitary gland functions normally in secreting other hormones [1]. The clinical features of CHH include incomplete or absent pubertal development and impaired or absent fertility in adulthood [1,2]. It can also be associated with non-reproductive system defects such as olfactory abnormalities, cleft lip and palate, and ear malformations. Olfactory loss can be observed in approximately 50% of CHH cases [3,4], and this co-occurrence is referred to as Kallmann’s Syndrome (KS), while CHH without olfactory impairment is called normosmic congenital hypogonadotropic hypogonadism (nCHH). The overall prevalence of CHH is 1/100,000 to 1/1,000,000, with a male-to-female ratio of 5:1 [5]. CHH is caused by genetic defects that affect neuron development and the GnRH signaling pathway, and approximately 50% of cases can be explained by genetics [1]. Currently, over 50 genes have been found to be associated with CHH in humans, including ANOS1 (anosmin 1), PROKR2 (prokineticin receptor 2), PROK2 (prokineticin 2), CHD7 (chromodomain helicase DNA binding protein 7), TACR3 (tachykinin receptor 3), TAC3 (tachykinin precursor 3), and others [2].

The diagnosis of CHH in patients should consider factors such as age and hormone levels. The domestic guidelines recommend that in males, after excluding other secondary causes, the diagnosis of CHH can be considered if there is a lack of development of secondary sexual characteristics and increased testicular volume after the age of 18, testosterone level ≤100 ng/dL, and low or “normal” levels of Follicle-Stimulating Hormone (FSH) and luteinizing hormone (LH). As an exclusive disease, CHH shares similar clinical features and hormone levels with Constitutional Delay of Growth and Puberty (CDGP) in early adolescence. Genetic mutations in genes such as PROKR2 and KISS1R (kisspeptin-1 receptor) can also be detected in CDGP patients [6]. CDGP patients tend to be thin and have slow growth, and they remain short with immature secondary sexual characteristics during puberty, while CHH patients exhibit features such as cryptorchidism, micropenis, prepubescent voice, olfactory impairment, hypospadias, and renal structural abnormalities. CHH patients have delayed bone age and lack a significant increase in height, but in adulthood, they often exhibit tall stature with arm length exceeding height [7]. CDGP patients typically initiate spontaneous puberty around the age of 18, which can be used to differentiate them from CHH. Previous studies have shown that in CDGP patients, the peak LH value (at 60 minutes) after a Luteinizing Hormone-Releasing Hormone (LHRH) test is >4 U/L, which can be used to differentiate CHH from CDGP. Additionally, CDGP patients can spontaneously initiate puberty after low-dose testosterone induction [8], a presentation similar to that of CHH patients with reversal, although the duration of induced puberty in CDGP patients is shorter.

For diagnosed CHH patients, treatment should be initiated immediately to induce the development of secondary sexual characteristics and prevent psychological abnormalities. Currently, common treatment options include low-dose testosterone/estrogen, Human Chorionic Gonadotropin (HCG)/HCG combined with human Menopausal Gonadotropin (hMG), and GnRH pumps. It is important to note that LH stimulates the secretion of endogenous testosterone by testicular Leydig cells and maintains spermatogenesis, while testosterone replacement therapy can suppress LH secretion and impair the process of spermatogenesis. Therefore, during testosterone replacement therapy, there will be no increase in testicular volume or the occurrence of sperm [9], and when testicular volume increases, the possibility of reversal of CHH should be considered [10]. Important indicators for determining the reversal of the hypothalamic-pituitary-gonadal axis include an increase in endogenous testosterone levels to the normal range, an increase in testicular volume, and the occurrence of spontaneous sperm production after discontinuing treatment.

To date, PROKR2 gene mutations have been detected in several cases of CHH with reversal, but the relationship between these mutations and reversal remains unclear. To further explore the correlation between this gene and the reversal of gonadal function, this study reports a case of CHH patient who exhibited spermatogenesis without the use of gonadotropin therapy. A missense mutation (c.337T>C) in the PROKR2 gene was identified through whole-exome sequencing, and phenotype and genotype analysis were performed on this patient.

Case presentation

The patient, a 35-year-old male, initially sought medical care 17 years ago (at 18 years of age, the year 2005) due to “lack of secondary sexual characteristics”. It was found that the patient had not experienced voice deepening, lacked Adam’s apple, axillary and pubic hair growth, and had underdeveloped testes and penis. A diagnosis of “delayed puberty” is suspected, and the patient received HCG treatment (2000 IU, twice a week) for 3 years, followed by testosterone therapy, which gradually initiated the development of secondary sexual characteristics and sperm production. Even during intermittent periods of testosterone treatment interruption over the past 6 years (with the longest gap being 9 months), the patient could naturally produce sperm and engage in sexual activity. His spouse conceived naturally four times, with two resulting in miscarriages. The patient’s parents were healthy, not closely related, and there was no family history of similar diseases. The patient was born at full term without complications, had a normal feeding history, no history of head trauma, and had normal olfactory and auditory sensitivity. He performed well academically. Currently, the patient’s height is 183.5 cm, weight is 71 kg, and fingertip-to-fingertip span is 192.5 cm. Laboratory test results showed no abnormalities in blood routine, liver and kidney function, electrolytes, or cortisol levels. Hormone levels were as follows: (at 18 years old) testosterone 35 ng/dL; (at 21 years old) testosterone 64 ng/dL, LH 1.09 IU/L, FSH 1.31 IU/L; (at 28 years old) GnRH stimulation test: FSH 0 min 2.14 IU/L, FSH (60 min) 4.82 IU/L, FSH (120 min) 6.53 IU/L; LH 0 min 1.89 IU/L; LH (60 min) 12.20 IU/L; LH (120 min) 14.43 IU/L; (at 34 years old, 9 months after discontinuing medication) hormone levels: Testosterone 284 ng/dL, estradiol <10.0 pg/mL, FSH 3.69 IU/L, LH 2.55 IU/L. Testicular ultrasound at 18 years old showed testicular sizes of 2.7 cm x 1.2 cm (right) and 2.1 cm x 1.0 cm (left). Semen analysis at 26 years old indicated a sperm density of 52.1 million/mL, PR% 26.0%, and NP% 11.0%. No abnormalities were observed in the pituitary MRI. Chromosome karyotyping revealed a G-banded karyotype of 46, XY. Whole exon sequencing identified a c.337T>C: p.Y113H missense mutation in the PROKR2 gene. Computer analysis using Polyphen-2, M-CAP, Mutation Taster, and other software suggested that this gene mutation could be the causative mutation in this patient.

Discussion

This study reports a case of Congenital Hypogonadotropic Hypogonadism (CHH) with the PROKR2 gene c.337T>C missense mutation, in which the patient exhibited gonadal function reversal after 3 years of HCG treatment and long-term testosterone therapy. In this study, we aimed to further investigate the factors contributing to gonadal function reversal in this patient by conducting genetic testing and pathogenicity analysis of the detected genes. Through whole exon sequencing, we identified a missense mutation (c.337T>C:p.Y113H) in the PROKR2 gene in our patient. Previous studies have shown that this gene mutation significantly reduces the cell surface expression of the prokineticin receptor 2 [11]. In functional studies, the Y113H PROKR2 mutation located in the extracellular region of the receptor has been associated with reduced expression, decreased ability to activate the MAPK pathway, and decreased intracellular calcium mobilization [12]. This mutation is extremely rare in the population, with a reported mutation frequency of 0.00146787 in the gnomAD database and a frequency of 0.000140003 in the Human Exome Database (ExAC). Previous studies have shown that the mutated amino acid is highly conserved across representative species [13], and the mutation rate of PROKR2:p.Y113H missense mutation in 200 normal controls is 1% (2/200). Additionally, we performed computer analysis using software such as Polyphen-2, PROVEAN, and Mutation Taster, which indicated that this gene mutation is likely a pathogenic factor in this patient. Therefore, according to the ACMG classification criteria [14], the PROKR2 gene c.337T>C mutation is considered to have possible pathogenic significance. This mutation has been reported as a Pathogenic variant (PS1) with a very low frequency (PM2), and it has been predicted to be deleterious and pathogenic by SIFT, PolyPhen-2, and Mutation Taster (PP3).

The Prokineticin receptor 2 (PROKR2) is a G-protein-coupled receptor expressed on the membrane of GnRH neurons and serves as a key regulatory factor in GnRH neuron development, olfactory development, and GnRH release. The PROKR2 gene is located on chromosome 20p13 and consists of two exons and seven transmembrane domains [15]. When PROKR2 binds to its ligand, prokineticin (PROK), it can activate multiple signaling pathways, including IP3/Ca2+, MAPK, and cAMP pathways, thereby promoting GnRH secretion. PROKR2 gene mutations are typically inherited in an autosomal dominant manner, but a few cases of autosomal recessive inheritance have also been reported, including heterozygous, homozygous, or compound heterozygous mutations [3,8], which is consistent with our study. The PROKR2 gene has been found to exhibit digenic and trigenic modes of inheritance, such as PROK2/PROKR2, FGFR1/PROKR2, PROK/GNRHR, and PROKR2/CHD7/FEZF1 [13,16,17]. In patients with normosmic congenital hypogonadotropic hypogonadism (nCHH) or Kallmann syndrome (KS), there are more than 27 genetic variations identified in the PROKR2 gene. PROKR2 gene mutations not only lead to KS but also contribute to nCHH, with clinical phenotypes ranging from partial pubertal development to complete absence of puberty, and some patients may exhibit undetectable LH levels, highlighting the high clinical heterogeneity of CHH. Non-reproductive phenotypes associated with CHH include diabetes, epilepsy, fibroplasia, and insomnia [18]. Although PROKR2 gene mutations exhibit variable reproductive phenotypes, non-reproductive phenotypes are relatively rare. Sinisi et al. [4,2] first observed the reversal phenomenon in a Kallmann patient with a homozygous PROKR2 gene mutation (Val274Asp), where after 5 years of gonadotropin and testosterone treatment, testosterone levels remained within the normal range even after 2 years of discontinuation. Sidhoum et al. [18] found no significant differences in the occurrence of cryptorchidism, micropenis, or partial pubertal development between patients with reversal and those without reversal in CHH. The spectrum of PROKR2 mutations in Chinese CHH patients has not yet been determined, but functional loss-of-function mutations in PROKR2 account for approximately 5% of CHH cases with or without olfactory impairments [15]. Due to the relatively small sample size in this study, the correlation between PROKR2 mutations and reversal cannot be determined, and further exploration is warranted.

Conclusion

After receiving hCG and long-term testosterone treatment, the patient experienced a recovery of gonadal axis function. Based on the diagnostic criteria for gonadal function reversal in Congenital Hypogonadotropic Hypogonadism (CHH), this papatient met the criteria for CHH gonadal function reversal. Whole exon sequencing detected a missense mutation (c.337T>C) in the PROKR2 gene in this patient. Bioinformatics analysis and ACMG guideline analysis confirmed that the PROKR2 gene c.337T>C missense mutation was a potential pathogenic variant. However, our statistical analysis indicated that this missense mutation in the PROKR2 gene could not fully explain the gonadal function reversal observed in this patient. It is important to note that CHH caused by genetic factors exhibits a high degree of clinical heterogeneity, and the presence of polygenic or oligogenic forms does not necessarily worsen the function of the hypothalamic-pituitary-gonadal axis in patients.

Declarations

Availability of data and materials: Not Applicable

Competing interests: The authors declare that they have no competing interests

Funding: Not applicable

Authors’ contributions: Not applicable

Acknowledgements: Not applicable

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