DOI: https://doi.org/10.21203/rs.3.rs-1717887/v1
Precocious puberty (PP) is defined as the onset of puberty in early childhood, which presents with testicular enlargement before 9 years in boys, and breast budding and the onset of menstruation before 8 years in girls. Hypothalamic hamartoma (HH) is among the most common cause of central PP (CPP). As the pathogenesis of HH has not been elucidated to date, we explored the underlying molecular changes in three HH cases encountered in order to provide clues for clinical treatment. All specimens were reviewed from a pathological standpoint and then checked using next-generation sequencing to elucidate gene mutation profiles. In all three cases with HH, the lysine-specific methyltransferase 2C (KMT2C/MLL3) missense mutations were found. Combined with its close relationship with estrogen-related cancers, we speculate that KMT2C is involved in the pathogenesis of CPP owing to the resultant changes in estrogenic levels and release of gonadotropin-releasing hormone.
Precocious puberty (PP) is defined as the onset of puberty in early childhood. It is divided into central PP (CPP) and peripheral PP (PPP) [1]. The main manifestation of PP is testicular enlargement before 9 years of age in boys, and breast budding and the onset of menstruation in girls before the age of 8 years. The pathophysiological basis for PP is the early activation of pulsatile gonadotropin-releasing hormone (GnRH) secretion, resulting from hypothalamic or neoplastic lesions (CPP or gonadotropin-dependent PP) [2]. The cause of PPP in most boys is attributed to chorionic gonadotropin-related tumors and is unknown in girls. Previous studies have shown that about 75% of the cases of CPP that occur before 3 years of age are due to hypothalamic hamartomas (HHs) [3]. HH presentations include endocrine or neurological abnormalities such as cognitive impairment and seizures, particularly gelastic epilepsy [4]. Previous studies have shown that some patients have no symptoms. The molecular pathogenesis of HH has not been elucidated to date. In this study, three patients with HH were identified and representative samples from them were collected and sequenced in an attempt to elucidate the molecular pathogenesis of HH.
Three cases of HH were diagnosed at Zhongnan Hospital of Wuhan University from 2016 to 2021. Specimens from all patients were obtained from resections. This study was approved by Research Ethics Committee of Zhongnan Hospital of Wuhan University.
The case 1 was born vaginally after 38 + 4 weeks of gestation. Early genital development, acne and erection were observed at 7 months age, that is the penis and testicles were enlarged, and pubic hair was present (Tanner stage: P3) (Supplementary data 1). MRI of the hypothalamus and pituitary gland showed a left posterior tubercle of the optic chiasma, measuring 80×70×20 mm (Supplementary data 2). Bone age was equivalent to that at 3 years of age (Supplementary data 2). No abnormalities were detected using echocardiography and chest radiography. Hyperactivity was found but without gelastic epilepsy or abnormalities in cognitive development.
Serum growth hormone (GH) levels were significantly elevated before operation, but did not return to normal follow up 6 months after resection.
The case 2 was a male born at full-term. The birth weight, length and head circumference were normal. Early genital development and the penis and testicles were enlarged compare than the peers at 21 months age. At the same time, acne and erection were observed. Hyperactivity and gelastic epilepsy were found but without abnormalities in cognitive development. Radiologically showed that lesions were located in the suprasellar region of hypothalamus and there was no enhancement in the brain. The lesions were subjected to total resection. Serum prolactin (PRL) and cortisol (CORT) levels were significantly elevated before operation, but did not return to normal follow up 17 months after resection (Table-1).
The case 3 was a female born at full-term. The girl's developmental indicators were normal until breast budding and menstrual cramps at six years old. Hyperactivity and gelastic epilepsy were found but without abnormalities in cognitive development just like case 2. Radiologically showed that lesions were located in the hypothalamus and arachnoid cyst of hypothalamus and there was no enhancement in the brain. The lesions were subjected to total resection. GH level increased significantly before surgical resection, but the patient lost follow-up after surgical resection 6 month and the hormone levels were unknown (Table-1).
Microscopically, the lesions of three cases were mainly composed of mature neurons and glial cells but showed an abnormal distribution pattern. In the exemplified HH case, the lesions were predominantly neuronal in composition and were diffusely distributed, with astrocytes comprising the main components of glia. Oligodendrocytes were rarely identified. The neurons were mostly small to medium in size (Figure-1 A and B). The background stroma of HH was composed of slightly vacuolated neuronal and glial processes similar to the normal counterpart of the central nervous system (Figure-1 B). NeuN highlighted the neurons within the nodules of HH, whereas neurofilament protein (NF) outlined the processes (Figure-1 C and F). Mature glial cells were positive with glial fibrillary acidic protein (GFAP) and oligodendrocyte-2 (oligo-2) (Figure-1 E and D).
The molecular pathology detection of the three cases for high-throughput sequencing. Coverage for all 550 genes exon region, and part of the 21 introns area (often happen FUSION area), to detect mutations in the form of point mutations (SNV), loss of small fragments inserted (INDEL), copy number variation (CNV) and FUSION.
The KMT2C/MLL3 gene located on chromosome 7q36 belongs to the TRX/MLL families of genes and the encoded protein belongs to ASCOM [5]. In all three cases of HH, KMT2C missense mutations were found to be NM_170606.2 exon18 c.2917A > G p.R973G. That said, nucleotide “A” at position 2917 on exon 18 was replaced by nucleotide “G,” resulting in the arginine “R” at position 973 in the corresponding protein sequence being replaced by the glycine “G.” The frequency of this mutation was 16.87%. Studies have shown that epigenetic regulators KMT2C/MLL3, which are mutated in > 50% of hepatocellular carcinomas, were also mutated in liver metastases. However, KMT2C missense mutations have not been reported in brain tumors.
In addition, IRS2 missense mutation NM_003749.2 exon1 c.3238G > A p.A1080T was detected in one of the three cases. The nucleotide G at position 3238 on exon 1 was replaced by nucleotide A, resulting in the alanine (A) at position 1080 of the corresponding protein sequence being replaced by threonine (T). The frequency of this mutation was 1.19%. IRS2 is a signal ligand in metabolism that encodes an intracellular signaling molecule to regulate insulin-like growth factor 1 and other cytokines and participates in the phosphorylation of insulin signal transduction. It is expressed in cancer cells and promotes the occurrence and metastasis of cancers, including breast cancer, esophageal cancer, colorectal cancer, and stomach cancer. The other less frequently mutated gene FCGR3A was detected, but the frequency of this mutation was very low.
No MSI was found in any of the three cases.
HH is a rare, congenital mass or lesion comprising mature neurons and glial cells but with an abnormal distribution[5]. However, the cytologically normal small neurons of HH are readily identified as opposed to the large pyramidal neurons of the normal hypothalamus or pyramidal-type neurons seen in ganglioglioma[6]. Moreover, the glial components of HH are normal and can be readily distinguished from other tumors. The pathogenesis of HH resulting in CPP remains to be verified because conflicting results have been reported in the literature [7].
An interesting finding of the present study is that KMT2C gene mutation was detected in the tissue samples of all three patients. Here, we attempted to elucidate its underlying relationship with PP. Although the location of HH lesion causing CPP has been well reported, the molecular mechanism in the early onset of puberty has not been defined. In the presence of GnRH, the pulsating secretion of pituitary gonadotropic hormone is a necessary condition for the onset of puberty. GnRH secretion in the human hypothalamus is regulated by kisspeptin, neurokinin B, dynorphin, and their corresponding receptors [8]. Estrogen is also involved in the release of GnRH, leading to the development of PP. In a comparative study that enrolled 73 patients with CPP and 101 normal adolescent girls, Rea gene (estrogen receptor) polymorphisms were found to be associated with early menarche [9]. Another study has identified eight polymorphisms in the ER alpha gene, which is a ligand-activated nuclear receptor that directs proliferation and differentiation in cancer cells including mammary cancer cells [10]. These findings suggest that increased estrogen receptor sensitivity may be caused by mutation or polymorphism of the ER alpha gene, which is related to the etiology of PP. Regulatory functions of ER alpha require a stable genomic environment [11]. KMT2C is required for ER alpha activity and breast cancer proliferation [11]. KMT2C gene mutation negatively regulates the expression of estrogen-dependent genes, and its deficiency can impair estrogen-driven breast cancer proliferation and promote tumor growth. Thus, KMT2C is one of the most commonly mutated genes in ER-positive breast cancer [12]. As discussed, we found KMT2Cmutations in all three patients and the upregulation of estrogen in one of the patients post-surgery (could not be determined in the other two patients). Based on the knowledge that theKMT2C gene is closely related to estrogen and its corresponding GnRH release; we speculated that KMT2C is implicated in the occurrence of CPP through altering the levels of estrogen.
Compared with the GLI3 gene reported involved in Pallister-Hall syndrome, which is occasionally associated with HH, the KMT2C gene is close to it spatially. The KMT2C gene is on the chromosomesame as GLI3 gene. However, no GLI3 mutation was identified in any of the patients in our study, indicating the likely involvement of other pathways. Further studies including the enrollment of more cases, experimental tests, and the use of animal models are necessary to elucidate the mechanism and to verify our hypothesis.
Funding support:
This work was supported by Zhongnan Hospital of Wuhan University Science, Technology and Innovation Seed Fund
Table-1 General information of the three cases
|
Case |
Age |
Gender |
Appearance characteristics |
MRI |
Hormone (pre-operation) |
Mutation |
Treatment |
Follow up time |
Hormone (post-operation) |
Symptoms |
|
Case 1 |
13 months |
male |
Early genital development; Erection; Acne |
Tumor in left posterior tubercle of optic chiasma |
GH↑ |
KMT2C; IRS2 |
Resection |
6 months |
GH↑ E2↑ |
Remission |
|
Case 2 |
21 months |
male |
Gelastic epilepsy; Testicular enlargement |
Tumor in suprasellar region |
PRL↑ CORT↑ |
KMT2C |
Resection |
17 months |
PRL↑ CORT ↑ |
Remission |
|
Case 3 |
6 years |
female |
Breast budding; Menstrual cramps |
Tumor in hypothalamus and arachnoid cyst |
GH↑ |
KMT2C |
Resection |
6 months |
- |
Remission |
GH, growth hormone; PRL, prolactin; CORT, cortisol; E2, estradiol