Clinical and Genetic Mechanisms in Patients with MC2R Deficiency Presenting with Early Puberty

Abstract

Introduction

Melanocortin receptor 2 (MC2R) in the adrenal cortex controls the hypothalamic-pituitary-adrenal axis. The melanocortin system, influenced by leptin, regulates GnRH neurons, crucial for puberty onset and fertility. This study evaluates early puberty in primary adrenal insufficiency (PAI) patients due to MC2R gene alterations.

Methods

Seven patients with PAI (P1-P7) from five unrelated families, all presenting with early or precocious puberty, were included. MC2R deficiency diagnosis ranged from 1 day to 11 months. MKRN3, DLK1, KISS1, and KISS1R genes were analyzed using Sanger sequencing in four cases (P2, P4, P6, and P7). All clinical data were obtained retrospectively.

Results

Puberty onset mean age was 8.6 years (7.4–9.5) in boys (P1, P2, P3, P7) and 8.5 years (7.4–9.5) in girls (P4, P5, P6). Tumor markers were negative; no adrenal rest or tumors were found. GnRH analogs were used for rapid puberty in P2, P3, P6. Final height in P1 and P2 was below target (−2.6 SDS, −0.7 SDS). Menarche occurred at 11 and 11.3 years in P4 and P5. No pathogenic variants were found.

Conclusion

Genetic causes of early puberty were not identified. Elevated ACTH may stimulate kisspeptin neurons, triggering puberty. Close monitoring of these patients for pubertal development is recommended.

Introduction

Puberty is a complex phenotypic trait regulated by the interplay of various genetic, environmental, and epigenetic factors [1]. Disruption of this network of pubertal regulation leads to defects in the timing of puberty such as central precocious puberty (CPP). CPP is defined as the development of secondary sexual characteristics before the age of 9 in boys and 8 in girls [2]. The mechanism of CPP remains largely unclear and the monogenic causes represent less than 15% of all, with the maternally imprinted MKRN3 gene being the most prevalent [3, 4].

DLK1 also inhibits the delta-notch pathway, significantly affecting both the reproductive and metabolic systems. Deficiency in DLK1 represents a rare monogenic etiology contributing to familial CPP [5, 6]. Rare, isolated instances of KISS1 gene variations have also been implicated in CPP [7].

Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive condition characterized by isolated glucocorticoid deficiency with normal mineralocorticoid function. Defects in the MC2R gene cause FGD-1 [8]. Some patients with pathogenic MC2R variations reported had tall stature; however, no definitive mechanism has been identified to date [9]. In addition, knowledge about the initiation of puberty and factors affecting pubertal progression in FGD-1 patients is limited.

Here, we report the clinical and molecular characteristics of 7 patients carrying loss-of-function MC2R alterations, all with precocious/early puberty. We aimed to investigate the pubertal status of these cases and explore the mechanisms influencing pubertal timing.

Patients and Methods

Seven patients with FGD-1 due to pathogenic MC2R variations were included in this study. This study was conducted retrospectively, and all data were obtained from medical records.

Patients

Height and weight were measured in all subjects and their parents using a wall-mounted calibrated Harpenden Stadiometer (Holtain Ltd, Crymych, UK) sensitive to 0.1 cm and an electronic scale sensitive to 0.1 kg. Body mass index (BMI) was calculated as the weight in kilograms divided by the height in meters squared. The standard deviation score (SDS) of all auxological measurements was calculated according to national data [10]. Target height (TH) was calculated using the formula, maternal height + paternal height ±13 cm/2. The onset of puberty was defined as breast Tanner stage 2 in females, and testicular volume of 4 mL in males. Bone age was evaluated by the Greulich and Pyle method. The predicted adult height was calculated using the Bayley Pinneau method [11]. Pelvic ultrasound and hypothalamic-pituitary MRI were used as additional diagnostic tools for the etiology of early puberty.

Molecular Genetic Analyses

The molecular techniques used for the analysis of the MC2R gene were previously described and identified variants are shown in Table 1 [12]. Genomic DNA samples were extracted from peripheral blood using standard protocols and the coding regions of the MKRN3 (NM_005664.3), DLK1 (NM_003836.6), KISS1 (NM_002256.3), and KISS1R (NM_032551.4) genes were sequenced using the Sanger sequencing method in P2, P4, P6, and P7. Specific primers were designed to cover all coding sequences and the exon-intron boundaries (primer sequences can be provided upon request). BigDye^® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher, USA) was used according to the instructions of the manufacturer. P1 was lost in follow-up due to sepsis-related multi-organ failure, hence genetic testing could not be performed in this case.

Table 1.

Clinical, laboratory, and molecular findings of patients

	Family 1	Family 2		Family 3	Family 4		Family 5
Patients	P1a	P2b	P3b	P4	P5b	P6b	P7
At presentation
Gender	Male	Male	Male	Female	Female	Female	Male
Consanguinity	1st-degree	1st-degree		1st-degree	–		3rd-degree
Birth weight SDS	−0.1	0.3	1.2	0.7	−0.52	−1.6	−1.2
MAM, years	13	11.5		12	12		14
Mother height, cm/SDS	158.6/−0.8	165.5/+0.4		157.2/−1.0	154.2/−1.5		155.5/−1.3
Father height, cm/SDS	169/−1.2	172/−0.7		164.8/−1.9	164.2/−2.0		179.8/+0.6
TH, cm/SDS	170.3/−1.0	175.3/−0.2		154.5/−1.4	152.2/−1.86		174.4/−0.3
Age, months	2nd week	11	1st week	6.5	2	1st week	1st week
Weight, kg/SDS	3.2/−1.3	11.4/1.1	3.8/0.3	8.8/+1.2	4/–1.6	3.52/+0.1	2.5/–2.3
Height, cm/SDS	50/−0.8	80.7/1.7	49.9/−0.4	70.7/+1.3	56.5/−0.1	49.8/−0.2	48/−1.3
BMI, kg/m2/SDS	12.8/−1.1	17.5/+0.1	15.3/+0.9	17.6/+0.4	12.5/−2.3	14.2/+0.3	10.9/−2.3
ACTH, pg/mL (N:7.2–63.3)	958	>1,250	>1,250	194.1	>1,250	802	1,250
Cortisol, µg/dL	0.1	0	1.2	<0.01	0	0.1	0.2
PRA, ng/mL/h (N: 1.9–6.0)	NA	7.7	1.9	3.2	NA	NA	127
Glucose, mg/dL	17	15	39	40	31	35	40
Additional problems	Mental-motor retardation and autism	Seizure, secondary hypothyroidism, hydrocele on right testis	–	Hypoglycemic seizure, primary hypothyroidism	–	–	Hypoglycemic seizure
MC2R gene sequence variations; zygosity	c.560delT/p.(Val187Alafs*29); homozygous	c.560delT/p.(Val187Alafs*29); homozygous		c.560delT/p.(Val187Alafs*29); homozygous	Deletion; homozygous		c.697G>C/p.A233P; homozygous

SDS, standard deviation score; MAM, maternal age at menarche; BMI, body mass index; PAH, predict adult height; ACTH, adrenocorticotropic hormone; PRA, plasma renin activity; NA, not available.

^aAt age of 25, he was exitus due to sepsis-related multiple organ failure.

^bPatients were siblings.

Results

Clinical Reports

The age at initial assessment of the patients varied from the first postnatal day to 11 months. Consanguineous parentage was observed in all cases. Birth weights were appropriate for gestational age. Primary adrenal insufficiency (PAI) manifested as hypoglycemia, convulsions, and hyperpigmentation. The diagnosis was conclusively confirmed through molecular analysis in all patients, as previously documented in a nationwide cohort. In this study, the MC2R association was reported at a rate of 26% (n = 25) [12]. The clinical findings at initial evaluation and during follow-up of the 7 cases included in the study are summarized in Tables 1 and Table 2. Except for the siblings (P2 and P3; P5 and P6), there was no family history of early puberty in the families.

Table 2.

Clinical, laboratory, and molecular findings of patients on follow-up

	Family 1	Family 2		Family 3	Family 4		Family 5
Patients	P1a	P2b	P3b	P4	P5b	P6b	P7
At onset of puberty
Age, years	9.5	8.4	7.4	8.5	9.5	7.8	9
Weight, kg/SDS	28.5/−0.4	30.1/0.6	33.2/2.1	40.2/+1.9	36.1/+0.6	39.7/+2.3	48.3/+2.9
Height, cm/SDS	125.6/−1.5	128.3/−0.2	126.0/+0.6	140/+1.6	139.3/+0.4	137.5/+1.9	138.7/+1.6
BMI, kg/m2/SDS	18.1/−0.5	18.3/+0.9	20.9/+2.1	20.5/+1.5	18.6/+0.6	21/+1.8	25.1/+2.6
Pubertal stage (Tanner)	2	2	2	2	2	2–3	2
	TV 4/4 mL, PH1	TV 4/4 mL, PH1	TV 4/4 mL, PH1	Breast 2/2, PH1	Breast 2/2, PH1	Breast 2–3/2, PH1	TV 4/4 mL, PH1
Bone age, years	8	4–5	6	7.8	10	8.8	8
PAH, cm/SDS	173.7	–	–	179	161.6/–0.3	167.4/+0.7	191/+2.4
ACTH, pg/mL	1,250	5.15	147	1,250	472	1,866	791
Basal LH, mIU/mL	0.6	1.3	0.7	2.2	0.6	0.1	0.65
Basal FSH, mIU/mL	1.0	1.7	0.81	1.7	0.48	1.33	2.41
GnRH test peak LH/FSH, mIU/mL	6.8/2	13.1/4.5	4.13/3.93	NA	NA	17.7/8.79	5.1/4.5
Testosterone, ng/mL/E2, pg/mL	0.28	3.29	0.3	–	–	–	0.3
				9.9		17.6
Scrotum/pelvic US	TR: 2.9 mL	TR: 2.3 mL	TR: 1.4 mL	Uterus: 3.6 mL	Uterus: 3.3 mL	Uterus: 3 mL	TR: 1.4 mL
Testes volume or uterus/ovaries	TL: 3.9 mL	TL: 1.8 mL	TL: 1.2 mL	R ovary: 0.8 mL	R ovary: 2.9 mL	R ovary: 4.1 mL	TL: 1.6 mL
				L ovary: 1.0 mL	L ovary: 2.1 mL	L ovary: 2.8 mL
Treatment	HC (15 mg/m2/day)	HC (15 mg/m2/day)	HC (18 mg/m2/day)	HC (8 mg/m2/day)	HC (14 mg/m2/day)	HC (10 mg/m2/day)	HC (10 mg/m2/day)
		GnRHa (3.75 mg/28 days)	GnRHa (3.75 mg/28 days)			GnRHa (3.75 mg/28 days)
At last evaluation
Age, years	23.1	17.1	14.9	19.9	13.95	10.17	11.73
Weight/SDS	63.3/−0.9	69.3/−0.01	70.8/+0.8	79.9/+2.8	49.7/–0.5	42.7/+1.17	62.5/+1.74
Height/SDS	160.5/−2.5	170.8/−0.7	170.8/+0.13	165.1/+0.3	155.5/–0.8	150.5/+1.73	152.1/+0.48
BMI, kg/m2/SDS	24.6/+0.5	23.8/+0.4	24.3/+0.9	29.3/+2.5	20.6/0	18.9/+0.58	27.02/+1.9
Pubertal stage (Tanner)	5	5	5	5	5	4	4
	TV 25/25 mL, PH 5	TV 15/25 mL, PH 5	TV 25/25 mL, PH 5	Breast 5/5, PH5 regular menstrual cycle	Breast 5/5, PH5 regular menstrual cycle	Breast 4/4, PH1 menstrual cycle absent	TV 10–12/10 mL, PH 1
Bone age, years	18	18	NA	18	NA	NA	9.5
Additional problems	Mental-motor retardation and autism	Right testicular microlithiasis	Bilateral testicular microlithiasis	Primary hypothyroidism (dyshormonogenesis)	–	–	Bilateral testicular microlithiasis

SDS, standard deviation score; BMI, body mass index; PH, pubic hair; ACTH, adrenocorticotropic hormone; LH, luteinizing hormone; FSH, follicle stimulating hormone; E2, estradiol; HC, hydrocortisone; GnRHa, gonadotropin releasing hormone analog; LT4, l-thyroxine; NA, not available.

^aAt age of 25, he was exitus due to sepsis-related multiple organ failure.

^bPatients were siblings.

Family 1

Patient 1 (P1) is the second born to his parents. He was diagnosed with PAI in the postnatal second week, and hydrocortisone replacement was initiated. He was diagnosed with autism spectrum disorder and risperidone treatment was commenced at 3 years of age.

At the age of 9.5 years, accelerated growth velocity (7.1 cm/year) was remarkable. Bilateral testicular volumes were 4 mL. The peak LH value on the GnRH stimulation test was 6.8 mIU/mL, compatible with puberty. He was followed up without any medical intervention for early puberty. At the age of 14.7 years, puberty was completed and bone age reached 16 years. Final height was ultimately below the TH.

Family 2

Patient 2 (P2) was referred due to recurrent hypoglycemia and myoclonic convulsions at the age of 11 months. He had frequent hospitalizations due to infections such as sepsis, meningitis, and pneumonia. The developmental milestones were delayed. He suffered seizures during the neonatal period and phenobarbital treatment was administered.

At referral, he had generalized hyperpigmentation, macroglossia, low-set ears, and bilateral hydrocele, while the systemic examination was otherwise normal. The diagnosis of PAI was confirmed based on biochemical and hormonal parameters, with no mineralocorticoid deficiency. Hydrocortisone replacement therapy was initiated. Additionally, the patient had secondary hypothyroidism with a TSH concentration of 5.42 mIU/mL (range: 0.27–4.2 μU/mL) and a free thyroxine 7.9 pmol/L (range: 12–22 pmol/L). The pituitary MRI was normal.

During follow-up, the size difference between the right and left testis was remarkable, he underwent ultimately right and left hydrocelectomy, when he was 2 and 14 years old, respectively.

At the age of 8.4 years, the patient entered puberty. Due to the rapid progression of puberty, GnRH agonist (GnRHa) treatment was initiated at the age of 10 years. However, after 1 year of treatment, the patient became noncompliant and discontinued the GnRHa therapy. In the most recent examination, a persistent size difference between the right and left testis was observed, and scrotal ultrasonography revealed calcifications in the right testis.

Patient 3 (P3) was the younger brother of P2. In the first hours after birth, he was diagnosed with PAI when evaluated for hypoglycemia.

At the ages of 5 and 6.5 years, P3 was hospitalized due to pseudotumor cerebri. The use of acetazolamide effectively resolved the elevated intracranial pressure, and no neuro-surgical interventions were needed. Control cranial MRI was normal. Puberty began at the age of 7.4 years, with a notably accelerated growth velocity of 12.1 cm/year. GnRHa treatment was administered between the ages of 8.4 and 12.2 years. In the latest examination, P3 was at Tanner stage 4 of puberty. Bone age was 12.5 years, and bilateral testicular microcalcifications were detected on ultrasonography.

Family 3

Patient 4 (P4) was evaluated for hypoglycemia in the first hours after birth. She was investigated for inborn errors of metabolism but the tests were all normal. She presented at the age of 6.5 months for further evaluation of hypoglycemia and PAI was detected. In addition, the patient was diagnosed with secondary hypothyroidism, as evidenced by a TSH concentration of 6.57 mIU/mL (range: 0.27–4.2 μU/mL) and a fT4 10.2 pmol/L (range: 12–22 pmol/L).

At the age of 8.5 years, puberty started. She was followed up without any medical intervention for early puberty. Menarche occurred at the age of 11 years.

Family 4

Patient 5 (P5) was diagnosed with PAI at the age of 2 months, during the investigation for neonatal cholestasis. At the age of 9 years, her breast stage was 2, and accelerated growth velocity was remarkable. The hormonal and radiologic examination was compatible with CPP. Predicted adult height was higher than TH, so she was followed up without any medical intervention. Menarche occurred at the age of 11.5 years. The final height was within TH.

Patient 6 (P6) was the fourth-born child of her parents. After birth, she has been admitted to the NICU. She was diagnosed with PAI and subsequently started treatment with hydrocortisone. Breast development and accelerated growth velocity were noted during the routine visit at the age of 7.8 years. Despite the progression of breast development, no significant pubic hair development was observed. While hormonal and pelvic imaging examinations indicated CPP (Table 1), cranial MRI was normal. GnRHa treatment was initiated.

Family 5

Patient 7 (P7) was diagnosed with PAI during the investigation of hypoglycemia and generalized hyperpigmentation. On the routine visit at the age of 9 years, testicular volumes were 4 mL. The peak LH concentration was 5.0 and the peak follicle stimulating hormone concentration was 4.5 on the GnRH stimulation test, suggesting the onset of puberty. Bone age was 8 years. No signs of pubertal acceleration or bone age advancement were observed. No cranial abnormalities were detected. There was no necessity for any treatment aimed at pubertal suppression. However, despite the progression of testicular volume, there was no significant advancement in pubic hair development observed.

Molecular Results

The Sanger sequencing of the MKRN3, DLK1, KISS1, and KISS1R genes in the recruited patients did not identify any pathogenic variants.

Discussion

Here we report the clinical and molecular findings of 7 patients with bi-allelic loss-of-function MC2R variations from five unrelated families, by presenting details of clinical findings, particularly by highlighting early pubertal development and also investigating underlying mechanisms. Isolated glucocorticoid or ACTH resistance caused by MC2R alterations is often accompanied by symptoms such as hypoglycemia, hyperpigmentation, hyperbilirubinemia, and cholestasis [12–14]. However, it is not commonly associated with early puberty or puberty precocious.

While stimulating the synthesis of the steroid hormone cortisol in adrenal cortex cells, ACTH also stimulates the synthesis of androgens. The activation of MC2R is an important step in this process, enhancing androgen production. Indeed, as a result, the blockade of the MC2R pathway also interrupts the production of androgens as well [15], which suggests that it may lead to androgen deficiency or delayed puberty. Liu et al. [14] reported a male patient with larger than normal genitalia despite the low level of androgens; however, they did not associate this with early puberty and detail the patient’s hormonal profile.

In some patients with MC2R alterations, hypothyroidism has been reported [13, 16, 17]. In our study, hypothyroidism was detected in 2 patients. However, there was no significant elevation of TSH or hypothyroidism that could trigger early puberty in these patients.

The history of pseudotumor in P3 could also contribute to precocious puberty. However, the presence of early puberty in other patients suggested the possibility of an underlying coexisting condition.

In our series, when assessing maternal menarche age, only the mothers of P2 and P3 had slightly earlier menarche compared to the mothers of the other cases. Except for the siblings, there was no family history of early puberty in the families. The MKRN3, DLK1 KISS1, and KISS1R genes are most commonly associated with familial or sporadic CPP [2]. Variants or alterations in these genes can contribute to early pubertal development. In this study, we have investigated the possible genetic determinants of CPP/early puberty by sequencing these genes in patients with PAI due to a pathogenic variant in the MC2R gene. The occurrence of early puberty in both siblings from family 2 and family 4 suggests the possibility of an associated genetic alteration in these genes, indicating a potential relationship. However, we did not detect any pathogenic or likely pathogenic variants in the aforementioned genes. Therefore, we have ruled out the possibility of these patients having an underlying genetic cause directly related to CPP/early puberty or dual diagnosis.

It has been reported that in individuals with pathogenic bi-allelic MC2R variations, high ACTH levels lead to excessive stimulation of the MC1R cartilaginous growth plates or stimulation of estradiol synthesis. Consequently, despite cortisol deficiency, progression in bone age may be observed, and an increase in height due to the unopposed effects of growth hormone [18, 19]. On the other hand, in our cases, there was no significant advancement in bone age that could trigger puberty.

Early puberty has been previously reported in individuals carrying pathogenic NNT and DAX-1 variations, with elevated ACTH levels in male patients with these alterations being associated with testicular enlargement and gonadotropin-independent precocious puberty [20–23]. However, in our study, we observed CPP in male patients based on hormonal profiles, there was no evidence of adrenal rest/tumor in the testes. Additionally, we also detected early puberty in female patients. Therefore, we were unable to explain the occurrence of early puberty in female patients with the mechanism of elevated ACTH levels.

ACTH is released from the anterior pituitary gland through the secretion of CRH in the hypothalamic paraventricular nucleus cells [24]. Kisspeptin neurons are expressed in two hypothalamic regions, namely the preoptic area and the arcuate nucleus [25, 26].

Additionally, an animal study demonstrated an increase in MC3R expression in KNDy and GHRH neurons, which is crucial in reproduction, while MC3R deficiency resulted in puberty delay [27]. Ultimately, we hypothesized that increased ACTH production in patients with MC2R variations may affect the stimulation of kisspeptin neurons in the arcuate hypothalamus either via the leptin pathway or directly, leading to an increase in GnRH secretion by increasing the MC3R links and thereby triggering the onset of puberty. Further observations and animal studies are needed to determine whether this mechanism is also applicable to other forms of poor control PAI.

In conclusion, early/precocious puberty may develop in individuals with associated MC2R alterations. Therefore, these patients should be closely monitored regarding pubertal development. Further studies are needed to elucidate the underlying mechanism.

Statement of Ethics

Medical ethical approval was granted by the Local Medical Ethics Committee of Istanbul University (2022/879945). Written informed consent was obtained from the parents and legal guardians of the participants under the age of 18 in our study. Written informed consents were obtained from the patients for publication of the details of their medical care and any accompanying images.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This study was sponsored by the Turkish Society for Pediatric Endocrinology and Diabetes, which provided support for the purchase of laboratory materials used in the genetic analyses of the patients.

Author Contributions

E.K.O., Z.Y.A, Ş.P., F.D., and F.B. clinically characterized the patients. V.K. and Z.O.U. performed and analyzed the sequencing data and evaluated the results. E.K.O., Z.Y.A., and F.B. prepared the draft manuscript. All authors contributed to the study’s conception, design, and discussion of results and edited and approved the final manuscript.

Funding Statement

This study was sponsored by the Turkish Society for Pediatric Endocrinology and Diabetes, which provided support for the purchase of laboratory materials used in the genetic analyses of the patients.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.