Non-invasive prenatal diagnosis of a paternally inherited MEN1 pathogenic splicing variant

Context Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease caused by mutations in the tumor suppressor gene MEN1. The uncertainty of pathogenicity of MEN1 variants complexifies the selection of the patients likely to benefit from specific care. Objective MEN1-mutated patients should be offered tailored tumor screening and genetic counselling. We present a patient with hyperparathyroidism for whom genetic analysis identified a variant of uncertain significance in the MEN1 gene (NM_130799.2): c.654G>T p.(Arg218=). Additional functional genetic tests were performed to classify the variant as pathogenic and allowed prenatal testing. Design Targeted next generation sequencing identified a synonymous variant in the MEN1 gene in a 26-year-old male with symptomatic primary hyperparathyroidism. In silico and in vitro genetic tests were performed to assess variant pathogenicity. Results Genetic testing of the proband’s unaffected parents showed the variant occurred de novo. Transcript study showed a splicing defect leading to an in-frame deletion. The classification of the MEN1 variant as pathogenic confirmed the diagnosis of MEN1 and recommended an adapted medical care and follow-up. Pathogenic classification also allowed to propose a genetic counselling to the proband and his wife. Non-invasive prenatal diagnosis was performed with a personalized medicine-based protocol by detection of the paternally inherited variant in maternal plasmatic cell free DNA, using digital PCR. Conclusion We showed that functional genetic analysis can help to assess the pathogenicity of a MEN1 variant with crucial consequences for medical care and genetic counselling decisions.

Neuronal Menin Overexpression Rescues Learning and Memory Phenotype in CA1-Specific α7 nAChRs KD Mice

The perturbation of nicotinic cholinergic receptors is thought to underlie many neurodegenerative and neuropsychiatric disorders, such as Alzheimer’s and schizophrenia. We previously identified that the tumor suppressor gene, MEN1, regulates both the expression and synaptic targeting of α7 nAChRs in the mouse hippocampal neurons in vitro. Here we sought to determine whether the α7 nAChRs gene expression reciprocally regulates the expression of menin, the protein encoded by the MEN1 gene, and if this interplay impacts learning and memory. We demonstrate here that α7 nAChRs knockdown (KD) both in in vitro and in vivo, initially upregulated and then subsequently downregulated menin expression. Exogenous expression of menin using an AAV transduction approach rescued α7 nAChRs KD mediated functional and behavioral deficits specifically in hippocampal (CA1) neurons. These effects involved the modulation of the α7 nAChR subunit expression and functional clustering at the synaptic sites. Our data thus demonstrates a novel and important interplay between the MEN1 gene and the α7 nAChRs in regulating hippocampal-dependent learning and memory.

A Novel Isogenic Human Cell-Based System for MEN1 Syndrome Generated by CRISPR/Cas9 Genome Editing

Multiple endocrine neoplasia type 1 (MEN1) is a rare tumor syndrome that manifests differently among various patients. Despite the mutations in the MEN1 gene that commonly predispose tumor development, there are no obvious phenotype–genotype correlations. The existing animal and in vitro models do not allow for studies of the molecular genetics of the disease in a human-specific context. We aimed to create a new human cell-based model, which would consider the variability in genetic or environmental factors that cause the complexity of MEN1 syndrome. Here, we generated patient-specific induced pluripotent stem cell lines carrying the mutation c.1252G>T, D418Y in the MEN1 gene. To reduce the genetically determined variability of the existing cellular models, we created an isogenic cell system by modifying the target allele through CRISPR/Cas9 editing with great specificity and efficiency. The high potential of these cell lines to differentiate into the endodermal lineage in defined conditions ensures the next steps in the development of more specialized cells that are commonly affected in MEN1 patients, such as parathyroid or pancreatic islet cells. We anticipate that this isogenic system will be broadly useful to comprehensively study MEN1 gene function across different contexts, including in vitro modeling of MEN1 syndrome.

Morphological characteristics of pituitary adenomas in the phenocopy of multiple endocrine neoplasia type 1

BACKGROUND: Multiple endocrine neoplasia type 1 (MEN 1) is a rare autosomal dominant disorder caused by mutations in the MEN1 gene, which encodes the menin protein. If a patient has the MEN 1 phenotype in the absence of mutations in the MEN1 gene, the condition is classified as a phenocopy of this syndrome. Although significant progress has been made in understanding the function of menin, its role in the oncogenesis of the endocrine glands is still being elucidated. Due to its key role in physiological and pathological processes, the assessment of the menin expression can provide valuable information.AIM: to determine whether there are any differences in the expression of menin in the pituitary adenomas (PA) in patients with phenocopy of MEN 1 (phMEN 1) and genetically confirmed MEN 1 (gMEN 1) compared with their sporadic forms.MATERIALS AND METHODS: immunohistochemical assessment of the menin expression was carried out in PA of patients with gMEN 1, phMEN 1 and sporadic acromegaly (SA), surgically treated in 2008–2020. IHC was performed using antibodies to menin, PRL, GH, ACTH, FSH, TSH, Pit-1, T-box, ERA on previously prepared histological section.RESULTS: The study included 35 samples of PA: gMEN 1 — 9 samples, phMEN 1 — 12 (somatotropinomas + PHPT); CA — 14 samples. The patients were comparable by gender, adenoma size, and drug intake. The gMEN 1 group differed from phMEN 1 and SA by age (p = 0.0005). In patients with gMEN 1, the expression of menin varied from no staining (5/9) to intense cytoplasm staining. Cytoplasmic expression of menin was mainly present (11/12) in the phMEN 1. In the SA group, there was no staining in 1 case; nuclear expression was detected in 6/14 cases. The phMEN 1 group showed significantly higher cytoplasmic expression of menin than the gMEN 1 group (p = 0.006). The gMEN 1 group also differed from the SA group (p = 0.012). There were no statistically significant differences between the phMEN 1 and SA groups (p = 0.049).CONCLUSION: It was revealed that the menin expression, in general, is retained in phMEN 1 and SA groups, although with different localization in the cell structure (nucleus and / or cytoplasm). At the same time, the expression of menin varies greatly in patients with gMEN 1. According to the data obtained, it can be assumed that the pathogenesis of PA in phMEN 1 and SA may have similarities; however, there could be factors contributing to the appearance of several tumors of the endocrine glands in one person with phMEN 1. To understand this process, it is necessary to further study the genes associated with MEN 1, epigenetic factors, signaling pathways in which menin is involved.

Role of miR-24 in Multiple Endocrine Neoplasia Type 1: A Potential Target for Molecular Therapy

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominant inherited multiple cancer syndrome of neuroendocrine tissues. Tumors are caused by an inherited germinal heterozygote inactivating mutation of the MEN1 tumor suppressor gene, followed by a somatic loss of heterozygosity (LOH) of the MEN1 gene in target neuroendocrine cells, mainly at parathyroids, pancreas islets, and anterior pituitary. Over 1,500 different germline and somatic mutations of the MEN1 gene have been identified, but the syndrome is completely missing a direct genotype-phenotype correlation, thus supporting the hypothesis that exogenous and endogenous factors, other than MEN1 specific mutation, are involved in MEN1 tumorigenesis and definition of individual clinical phenotype. Epigenetic factors, such as microRNAs (miRNAs), are strongly suspected to have a role in MEN1 tumor initiation and development. Recently, a direct autoregulatory network between miR-24, MEN1 mRNA, and menin was demonstrated in parathyroids and endocrine pancreas, showing a miR-24-induced silencing of menin expression that could have a key role in initiation of tumors in MEN1-target neuroendocrine cells. Here, we review the current knowledge on the post-transcriptional regulation of MEN1 and menin expression by miR-24, and its possible direct role in MEN1 syndrome, describing the possibility and the potential approaches to target and silence this miRNA, to permit the correct expression of the wild type menin, and thereby prevent the development of cancers in the target tissues.

Tissue-Specific Tumorigenesis in Multiple Endocrine Neoplasia Type I

While a germline heterozygous mutation in the MEN1 gene predisposes tumor formation in the endocrine pancreas, parathyroid glands and anterior pituitary, this tissue-specific tumorigenesis is not dependent on MEN1 mutations alone. In fact, a homozygous deletion of Men1 in the entire pancreas of a mouse results in tumor formation only in the endocrine pancreas, not in the exocrine pancreas, suggesting an endocrine tissue-specific mechanism. The MEN1 gene encodes the menin protein, which interacts with chromatin associated protein complexes, therefore engaging in epigenetic control mechanisms. Recognizing menin’s participation in epigenetic regulation led to an investigation of whether the pathogenesis of MEN1 syndrome may be related to epigenetic changes in the affected endocrine tissues. Indeed, MEN1-associated endocrine cell types exhibit various menin-dependent epigenetic mechanisms. In fact, a significant increase in methylated DNA loci was observed in MEN1 human parathyroid tumors when compared to human parathyroid adenomas and carcinomas without known MEN1 mutations. Subsequent studies revealed that loss of menin results in increased activity of DNA methyltransferase 1 (Dnmt1). Our studies have shown that Dnmt1 is transcriptionally regulated by the menin-interacting protein Rbbp5. While menin normally functions to suppress Rbbp5 activity, loss of menin activates Rbbp5, thus upregulating Dnmt1 expression, causing global DNA hypermethylation and subsequent tumorigenesis in MEN1-target endocrine tissues. In order to assess the behavior of Rbbp5 in both MEN1-target tissues and non-target tissues, Rbbp5 protein expression was analyzed in both MEN1-target tissues (endocrine pancreas, anterior pituitary, parathyroid) and non-MEN1-target tissues (kidney, lung, liver, brain, heart) of wild-type (WT) mice. We confirmed that Rbbp5 protein expression is ubiquitous throughout all of these WT mouse tissues. Since Rbbp5 is a transcriptional activator responsible for enhanced Dnmt1 gene expression, and the loss of menin causes Dnmt1 overexpression solely in MEN1-target tissues, we assessed whether Rbbp5 binds preferentially in a tissue-specific manner to the Dnmt1 promoter. We determined the presence of Rbbp5 on the Dnmt1 promoter in MEN1-target tissues (WT mouse endocrine pancreas, normal human parathyroid, WT mouse pituitary) and the absence of Rbbp5 on the Dnmt1 promoter in non-MEN1-target tissues (WT mouse liver, WT mouse kidney, WT mouse lung). These results confirmed that Rbbp5 does exhibit MEN1-target-tissue-specific occupancy at the Dnmt1 promoter. This endocrine-specific localization of Rbbp5 to the Dnmt1 promoter suggests the presence of additional tissue-specific factors (with tissue-specific expression or interactions/activity) that must be validated and tested further.

Modern views on the genetic determinism of GH-secreting pituitary adenomas (literature review and own data)

Background. This article presents a review of the current literature on the role of the genetic component in the etiology and pathogenesis of hormone-active pituitary adenomas secreting growth hormone (GH) and clinically manifesting by acromegaly and/or gigantism (multiple endocrine neoplasia 1 (MEN-1), McCune-Albright syndrome, Carney complex, X-linked acrogigantism (X-LAG), familial isolated pituitary adenoma — FIPA). Materials and methods. To identify mutations in the AIP gene and to verify FIPA, 26 patients of the Ukrainian population (19 women and 7 men) were examined in whom acromegaly was diagnosed in adolescence or young age, and genetic analysis was performed. To determine the genetic determinism in the development of GH-secreting pituitary adenoma and differential diagnosis of FIPA and MEN-1 syndromes by sequencing method (MLPA — ligation-dependent probe amplification), the genes MLPA, P244-C1 were studied involving exons 1–6 MEN1 gene, (MLPA, P017-D1) AIP gene. Results. Among those examined, only two patients had AIP gene mutations. In one patient, genetic screening for MEN1 gene mutation was negative and no clinical symptoms suggestive of McCune-Albright syndrome were detected. A variant heterozygous missense c.714C>G (p.Cys238Trp) was found in the AIP gene. This AIP gene assay is compatible with a genetic predisposition to develop pituitary adenoma. The offspring of this patient has a 50% chance of inheriting this variant, acromegaly, hypersomatotropinemia, MEN-1 syndrome, familial isolated pituitary adenoma. Another patient was diagnosed with MEN syndrome type 1 (Wermer syndrome): insulinoma, parathyroid gland adenomas (2), primary hyperparathyroidism with a heterozygous c.134A>G variant (p.Glu45Gly) found in the MEN1 gene. The presence of the c.l34A>G (p.Glu45Gly) class variant 4 is likely to be pathogenic. The prevalence of this variant in the general population is unknown, so it is very rare. Conclusions. The genetic analysis is appropriate in pediatric and young patients or those with GH-secreting macro/giant pituitary adenoma diagnosed at a young age (under 35), regardless of family history. In patients with a history of a disease, genetic analysis is recommended in any case to identify FIPA and to predict the further course of the disease and the effectiveness of treatment with somatostatin analogues.

Clinical Profile and Mutations Associated with Multiple Endocrine Neoplasia-Type1 (MEN1) and Their First-Degree Relatives at Risk of Developing MEN1: A Prospective Study

Multiple Endocrine Neoplasia type-1 (MEN1) is an autosomal dominant disorder with a combined occurrence of tumours of parathyroid glands, pancreatic islets, and anterior pituitary. About 90% of these patients carry mutations in the MEN1 gene, though the spectrum is not well defined in India. Forty clinically suspected cases of MEN1 were enrolled prospectively over six years; 32 patients (23 index-cases and nine affected relatives) with≥2 classical endocrine tumours of MEN1 were considered definite, and eight were categorised as ‘MEN1-like’. Details of their clinical presentation, treatment and mutational analysis including MEN1 gene, 3′ and 5′ untranslated regions (UTR) of MEN1, CDKN1B, and CaSR genes were collated. Asymptomatic first-degree relatives were also screened. Among the 32 definite MEN1 patients, all had primary hyperparathyroidism, 22 (68.7%) had gastroentero-pancreatic neuroendocrine tumours, and 21 (66%) had pituitary adenoma. Of the 23 definite index-cases, 13 (56.5%) carried mutations in the MEN1 gene. Five of nine affected first-degree relatives (55.5%), and four of 10 asymptomatic relatives (40%) also had MEN1 mutations. Seven of 10 MEN1 mutation-negative definite index-cases harboured p.V109G polymorphism in the CDKN1B gene. All eight MEN1-like cases were negative for mutations and large deletions in MEN1, mutations in 3′ and 5′ UTR of MEN1, CaSR and CDKN1B genes. The study has helped to clearly document the pattern of mutations among Indian MEN1 patients. However, the absence of MEN1 mutation in ~44% of cases and the presence of p.V109G polymorphism in CDKN1B gene raise the question whether such polymorphisms could independently contribute to pathogenesis.