Letteratura scientifica selezionata sul tema "MKRN3"

AbstractPuberty is a critical period of development regulated by genetic, nutritional, and environmental factors. The role of makorin ring finger protein 3 (MKRN3) in the regulation of pubertal timing was revealed when loss-of-function mutations were identified in patients with central precocious puberty (CPP). To date, MKRN3 mutations are the most common known genetic cause of CPP. MKRN3 is a member of the makorin family of ubiquitin ligases, together with MKRN1 and MKRN2. The Mkrn genes have been identified in both vertebrates and invertebrates and show high evolutionary conservation of their gene and protein structures. While the existence of Mkrn orthologues in a wide spectrum of species suggests a vital cellular role of the makorins, their role in puberty initiation and endocrine functions is just beginning to be investigated. In this review, we discuss recent studies that have shown the involvement of Mkrn3 and other makorins in the regulation of pubertal development and other endocrine functions, including metabolism and fertility, as well as their underlying mechanisms of action.

Background. Central precocious puberty (CPP) is defined by gonadotropin-dependent development of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys. MKRN3 and DLK1 are two genes, disease-causing variants of which have recently been discovered to cause idiopathic CPP. Methods. We screened 173 Chinese patients (9 males and 164 females; 9 familial and 164 sporadic) with ICPP and 43 patients (9 males and 34 females; 3 familial and 40 sporadic) with early puberty for variants in MKRN3. We also screened 19 patients with ICPP and early puberty for variants of DLK1 (17 males and 2 females; 5 familial and 14 sporadic). Results. We identified four novel missense variants of MKRN3, c.1138G > A (p.Glu380Lys), c.1420T > A (p.Leu474Met), c.673C > G (p.Leu225Val), and c.1071C > G (p.Ile357Met) in two sporadic cases and three familial cases. According to ACMG standards, two MKRN3 variant (p.Glu380Lys and p.Ile357Met) are likely pathogenic, and two others are of uncertain significance. We also performed bioinformatic analysis to evaluate the impact of variants on MKRN3 protein structures, which showed that Ile357Met locates at the zinc-binding region (C3HC4 RING finger motif), while Glu380Lys is spatially extremely close to the C3HC4 RING finger, MKRN-specific Cys-His domain, and the third C3H1 zinc-finger motif region. Per Glu380Lys, Glu with negative charges has been changed into Lys with positive charges, which may affect the hydrogen bond formation between amino acids and the stability of the local structure, thus affecting the binding of zinc iron to MKRN3 protein. Besides, we did not identify any variants of DLK1 gene in our patients. Conclusions. In this study, we report four novel MKRN3 variants in patients with ICPP. Moreover, we did not find any variants of DLK1 gene. Variants of MKRN3 are relatively uncommon in Chinese ICPP patients.

Makorin ring finger proteins (MKRNs) are part the of ubiquitin-proteasome system; a complex system important for cell functions. Ubiquitin fate through proteolytic, non-proteolytic pathways varies, depending on covalent linkage between ubiquitin and protein substrates. Makorin ring finger protein 3 is an integral part of covalent linkage of ubiquitin to protein substrates. Similar to others imprinted genes, MKRN3 also evolve under positive selection; however, which codons are specifically selected in MKRN3 during evolution are needed to be explored. Different maximum-likelihood (ML) codon-based methodologies were used to ascertain positive selection signatures in 22 mammalian sequences of MKRN3 to probe an individual codon for positive selection signatures. By applying the HyPhy software package implemented in the Data Monkey Web Server and CODEML implemented in PAML, evolutionary analysis based on two Ml frameworks were conducted. The analysis was executed by comparing M1a against M2a, M7 against M8, and PAML models and 2∆Lnl ( LRT) was resulted by likelihood logs. M1a contributed ω1 ( dN/dS) with LRT value ( ∆Lnl) 12.01, and positive selection was found in M2a with ω3 = 2.23603. To further improve selection test, M8 was compared to M7 with 2∆ Lnl ( LRT) 30.17, and M8 showed positive selection with ω = 1.55759. The data were fit to M8 than M7, which suggests that M8 was the most significant model of selection. M8 was judged encouraging for this analysis and used to establish a positive selection of MKRN3 proteins. We found Gly312 as a positively selected amino acid in a zinc finger motif/Really Interesting New Gene (RING) finger motif; the former ones’ region is involved in RNA binding and the later ones in ubiquitin ligase activity of the protein, vital for protein function. Selection analyses of MKRNs might advance the developments in unique approaches that could lead to genetic progress over the selection of superior individuals with the breeding values higher for certain traits as ancestries to get the next generation.

Abstract Background and Objectives Human fertility is attained following puberty due to finely orchestrated events driven by hypothalamic release of the excitatory neuropeptide neurokinin B (NKB), which in turn stimulates the release of kisspeptin and then GnRH to activate the downstream pituitary-gonadal axis. Our group has identified that loss-of-function mutations in Makorin Ring Finger Protein 3 (MKRN3) cause central precocious puberty (CPP). Mkrn3 is highly expressed in the hypothalamus of both male and female mice in early postnatal life, then declines prior to the onset of puberty. Mutations in MKRN3 E3 ubiquitin ligase domain, identified in patients with CPP, result in reduced auto-ubiquitination. However, the mechanisms of action of MKRN3 and its targets remain largely unknown. We hypothesized that MKRN3 inhibits the reproductive axis through interactions with hypothalamic substrates, targeting them to degradation pathways. Methods and Results Using interactome analysis and candidate approaches in vitro, we identified poly(A) binding protein cytoplasmic 1 and 4 (PABPC1 and 4) and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) as MKRN3 interactors, consistent with reports from other research groups. Additionally, our proteomic analysis revealed that Igf2bp1 levels are increased in the hypothalamus of Mkrn3 knockout (KO) mice versus wild type animals. Remarkably, we found that MKRN3-IGF2BP1 interaction is mediated by RNA, as their interaction was abrogated by RNase treatment. However, an MKRN3 missense mutation associated with CPP, p.C364F, within the E3 ubiquitin ligase domain, did not affect the RNA mediated interaction. Preliminary data from RNA co-IP, aiming to identify putative RNA targets, suggested that MKRN3 and IGF2BP1 bind to TAC3 mRNA. We further identified an increase in Nkb protein levels in the hypothalamus of Mkrn3 KO mice. We also showed that NKB is a substrate of MKRN3 E3 ubiquitin ligase activity in vitro by demonstrating increased ubiquitination of NKB in cells co-transfected with expression vectors encoding MKRN3 and NKB and treated with proteasome inhibitor. Additionally, MKRN3 overexpression resulted in degradation of NKB protein by western blot analysis, and alteration of NKB intracellular localization by immunofluorescence. These effects were impaired by MKRN3 missense mutations (p.C340G and p.C364F) in the E3 ubiquitin ligase motif, suggesting a pathophysiological mechanism of MKRN3 mutations in CPP. Conclusions We identified that MKRN3 interacts with PABPC1, PABPC4 and IGF2BP1 and targets NKB to degradation. We showed that NKB degradation was mediated by the MKRN3 E3 ubiquitin ligase domain, as mutations within this motif abrogated NKB degradation. However, this mutation did not affect MKRN3 RNA-mediated binding to IGF2BP1, highlighting that multiple domains of MKRN3 may contribute to its actions. Here, we propose a mechanism by which MKRN3, interacting with PABPCs and IGF2BP1 and targeting NKB for degradation, may act to inhibit the reproductive axis. Presentation: Sunday, June 12, 2022 11:00 a.m. - 11:15 a.m.

Abstract Context Loss-of-function mutations in the imprinted genes MKRN3 and DLK1 cause central precocious puberty (CPP) but whole gene deletions have not been reported. Larger deletions of the chromosome 15q11-13 imprinted locus, including MKRN3, cause Prader-Willi syndrome (PWS). CPP has been reported in PWS but is not common, and the role of MKRN3 in PWS has not been fully elucidated. Objective To identify copy number variants in puberty-related, imprinted genes to determine their role in CPP. Methods Probands with idiopathic CPP had chromosomal microarray (CMA) and targeted deletion/duplication testing for MKRN3 and DLK1. Results Sixteen female probands without MKRN3 or DLK1 variants identified by Sanger sequencing were studied. Whole gene deletions of MKRN3 were identified in 2 subjects (13%): a complete deletion of MKRN3 in Patient A (pubertal onset at 7 years) and a larger deletion involving MAGEL2, MKRN3, and NDN in Patient B (pubertal onset 5.5 years). Both were paternally inherited. Patient B had no typical features of PWS, other than obesity, which was also present in her unaffected family. Conclusions We identified 2 cases of whole gene deletions of MKRN3 causing isolated CPP without PWS. This is the first report of complete deletions of MKRN3 in patients with CPP, emphasizing the importance of including copy number variant analysis for MKRN3 mutation testing when a genetic diagnosis is suspected. We speculate that there is a critical region of the PWS locus beyond MKRN3, MAGEL2, and NDN that is responsible for the PWS phenotype.

Context: Loss-of-function mutations in the coding region of MKRN3, a maternally imprinted gene at chromosome 15q11.2, are a common cause of familial central precocious puberty (CPP). Whether MKRN3 alterations in regulatory regions can cause CPP has not been explored to date. We aimed to investigate potential pathogenic variants in the promoter region of MKRN3 in patients with idiopathic CPP. Patients/Methods: A cohort of 110 patients with idiopathic CPP was studied. Family history of precocious sexual development was present in 25%. Mutations in the coding region of MKRN3 were excluded in all patients. Genomic DNA was extracted from peripheral blood leukocytes, and 1,100 nucleotides (nt) of the 5′-regulatory region of MKRN3 were amplified and sequenced. Luciferase assays were performed in GT1–7 cells transiently transfected with plasmids containing mutated and wild-type MKRN3 promoter. Results: We identified a rare heterozygous 4-nt deletion (c.-150_-147delTCAG; –38 to –41 nt upstream to the transcription start site) in the proximal promoter region of MKRN3 in a girl with CPP. In silico analysis predicted that this deletion would lead to the loss of a binding site for a downstream res­ponsive element antagonist modulator (DREAM), a potential transcription factor for MKRN3 and GNRH1 expression. Luciferase assays demonstrated a significant reduction of MKRN3 promoter activity in transfected cells with a c.-150_- 147delTCAG construct plasmid in both homozygous and heterozygous states when compared with cells transfected with the corresponding wild-type MKRN3 promoter region. Conclusion: A rare genetic alteration in the regulatory region of MKRN3 causes CPP.

Abstract Background There is no data regarding the interrelationships of circulating Makorin Ring Finger Protein-3 (MKRN3), Kisspeptin (KISS1), and Neurokinin B (NKB) concentrations during minipuberty in humans. Objective To determine temporal changes in circulating concentrations of MKRN3, KISS1, NKB, and gonadotropins and investigate interrelationships between them in healthy full-term (FT) and preterm (PT) infants during minipuberty period. Methods A prospective study of 6-month follow-up performed. Eighty-seven healthy newborns, 48 FT (19 boys/29 girls), and 39 PT (21 boys/18 girls) (gestational age 31–37 weeks), were included. Blood samples were taken at 7 days (D7), 2 months (M2), and 6 months (M6) of age. Serum MKRN3, KISS1, NKB, LH, FSH, total testosterone (TT), and estradiol (E2) concentrations were measured. Results Seventy infants completed the study. MKRN3, KISS1, and NKB concentrations were similar in FT girls and boys. PT boys and girls also had similar concentrations of MKRN3, KISS1, and NKB. FT babies had significantly higher NKB concentrations than PT babies at D7, M2, and M6. MKRN3 and KISS1 concentrations do not differ between FT and PT babies. A strong positive correlation was found between MKRN3 and KISS1 at each time point and in all groups. FSH, LH, TT/E2 concentrations decrease while those of MKRN3 and KISS1 have a trend to increase toward the end of minipuberty. No correlation was detected between gonadotropins and MKRN3, KISS1, NKB concentrations. Conclusion Strong positive correlation demonstrated between KISS1 and MKRN3 suggests that interrelationship between molecules controlling minipuberty is not similar to those at puberty.

Introduction: Loss-of-function mutation of MKRN3 represents the most frequent genetic cause of familial central precocious puberty (CPP). The outcomes of gonadotropin-releasing hormone analog (GnRHa) treatment in CPP patients with MKRN3 defects are unknown. Objective: To describe the clinical and hormonal features of patients with CPP with or without MKRN3 mutations after GnRHa treatment. Anthropometric, metabolic and reproductive parameters were evaluated. Patients and Methods: Twenty-nine female patients with CPP due to loss-of-function mutations in the MKRN3 and 43 female patients with idiopathic CPP were included. Their medical records were retrospectively evaluated for clinical, laboratory, and imaging study, before, during, and after GnRHa treatment. All patients with idiopathic CPP and 11 patients with CPP due to MKRN3 defects reached final height (FH). Results: At the diagnosis, there were no significant differences between clinical and laboratory features of patients with CPP with or without MKRN3 mutations. A high prevalence of overweight and obesity was observed in patients with CPP with or without MKRN3 mutations (47.3 and 50%, respectively), followed by a significant reduction after GnRHa treatment. No significant differences in the values of mean FH and target height were found between the 2 CPP groups after GnRHa treatment. Menarche occurred at the expected age in patients with or without CPP due to MKRN3 mutations (11.5 ± 1.3 and 12 ± 0.6 years, respectively). The prevalence of polycystic ovarian syndrome was 9.1% in patients with CPP due to MKRN3 mutations and 5.9% in those with idiopathic CPP. Conclusion: Anthropometric, metabolic, and reproductive outcomes after GnRHa treatment were comparable in CPP patients, with or without MKRN3 mutations, suggesting the absence of deleterious effects of MKRN3 defects in young female adults’ life.

Background: Recently, mutations of makorin RING finger protein 3 (MKRN3) have been identified in familial central precocious puberty (CPP). Serum levels of this protein decline before the pubertal onset in healthy girls and boys and are lower in patients with CPP compared to prepubertal matched pairs. The aim of our study was to investigate longitudinal changes in circulating MKRN3 levels in patients with CPP before and during GnRH analogs (GnRHa) treatment. Methods: We performed a longitudinal prospective study. We enrolled 15 patients with CPP aged 7.2 years (range: 2–8) with age at breast development onset < 8 years and 12 control girls matched for the time from puberty onset (mean age 11.8 ± 1.2 years). Serum values of MKRN3, gonadotropins, and 17β-estradiol were evaluated before and during treatment with GnRHa (at 6 and 12 months). The MKRN3 gene was genotyped in CPP patients. In the girls from the control group, only basal levels were analyzed. Results: No MKRN3 mutations were found among CPP patients. MKRN3 levels declined significantly from baseline to 6 months of GnRHa treatment (p = 0.0007) and from 6 to 12 months of treatment (p = 0.003); MKRN3 levels at 6 months were significantly lower than in the control girls (p < 0.0001). Conclusions: We showed that girls with CPP had a decline in peripheral levels of MKRN3 during GnRHa treatment. Our data suggest a suppression of MKRN3 by continuous pharmacological administration of GnRHa.

ObjectiveMakorin ring finger protein 3 (MKRN3) gene restrains the hypothalamic–pituitary–gonadal axis. In girls, peripheral levels of MKRN3 decline prior to the onset of puberty. We described longitudinal changes in serum MKRN3 levels in boys before and during puberty and assessed the effect of inhibition of estrogen biosynthesis on MKRN3 levels.DesignLongitudinal serum samples from a double-blind, randomized controlled study in 30 boys (age range: 9.1–14.2years) with idiopathic short stature who received placebo (Pl;n=14) or aromatase inhibitor letrozole (Lz; 2.5mg/day;n=16) for 2years.MethodsWe analyzed the relationships between serum MKRN3 and clinical and biochemical markers of puberty by using summary measures.ResultsSerum MKRN3 declined by 669±713 pg/mL per year (P<0.001). This change was biphasic, as the levels decreased during Tanner genital stage G1 (–2931±2750 pg/mL per year) and plateaued thereafter (–560±1510 pg/mL per year) (P<0.05). During G1, MKRN3 levels in Lz-treated subjects decreased slower than in Pl-treated boys (–782±3190 vs –2030±821 pg/mL per year,P<0.05). The decrease in serum MKRN3 levels in G1 was associated with increases in LH (r=–0.5,P<0.01), testosterone (r=–0.6,P<0.01), and inhibin B (r=–0.44,P<0.05) (n=26).ConclusionPeripheral MKRN3 levels in boys appear to serve as a readout of the diminishing central inhibition that controls the onset of puberty.

A maioria dos casos de puberdade precoce central (PPC) em meninas permanece idiopática. A hipótese de uma causa genética vem se fortalecendo após a descoberta de alguns genes associados a este fenótipo, sobretudo aqueles implicados com o sistema kisspeptina (KISS1 e KISS1R). Entretanto, apenas casos isolados de PPC foram relacionados à mutação na kisspeptina ou em seu receptor. Até recentemente, a maioria dos estudos genéticos em PPC buscava genes candidatos selecionados com base em modelos animais, análise genética de pacientes com hipogonadismo hipogonadotrófico, ou ainda, nos estudos de associação ampla do genoma. Neste trabalho, foi utilizado o sequenciamento exômico global, uma metodologia mais moderna de sequenciamento, para identificar variantes associadas ao fenótipo de PPC. Trinta e seis indivíduos com a forma de PPC familial (19 famílias) e 213 casos aparentemente esporádicos foram inicialmente selecionados. A forma familial foi definida pela presença de mais de um membro afetado na família. DNA genômico foi extraído dos leucócitos do sangue periférico de todos os pacientes. O estudo de sequenciamento exômico global realizado pela técnica ILLUMINA, em 40 membros de 15 famílias com PPC, identificou mutações inativadoras em um único gene, MKRN3, em cinco dessas famílias. Pesquisa de mutação no MKRN3 realizada por sequenciamento direto em duas famílias adicionais (quatro pacientes) identificou duas novas variantes nesse gene. O MKRN3 é um gene de um único éxon, localizado no cromossomo 15 em uma região crítica para a síndrome de Prader Willi. O gene MKRN3 sofre imprinting materno, sendo expresso apenas pelo alelo paterno. A descoberta de mutações em pacientes com PPC familial despertou o interesse para a pesquisa de mutações nesse gene em 213 pacientes com PPC aparentemente esporádica por meio de reação em cadeia de polimerase seguida de purificação enzimática e sequenciamento automático direto (Sanger). Três novas mutações e duas já anteriormente identificadas, incluindo quatro frameshifts e uma variante missense, foram encontradas, em heterozigose, em seis meninas não relacionadas. Todas as novas variantes identificadas estavam ausentes nos bancos de dados (1000 Genomes e Exome Variant Server). O estudo de segregação familial em três dessas meninas com PPC aparentemente esporádica e mutação no MKRN3 confirmou o padrão de herança autossômica dominante com penetrância completa e transmissão exclusiva pelo alelo paterno, demonstrando que esses casos eram, na verdade, também familiares. A maioria das mutações encontradas no MKRN3 era do tipo frameshift ou nonsense, levando a stop códons prematuros e proteínas truncadas e, portanto, confirmando a associação com o fenótipo. As duas mutações missenses (p.Arg365Ser e p.Phe417Ile) identificadas estavam localizadas em regiões de dedo ou anel de zinco, importantes para a função da proteína. Além disso, os estudos in silico dessas duas variantes demonstraram patogenicidade. Todos os pacientes com mutação no MKRN3 apresentavam características clínicas e hormonais típicas de ativação prematura do eixo reprodutivo. A mediana de idade de início da puberdade foi de 6 anos nas meninas (variando de 3 a 6,5) e 8 anos nos meninos (variando de 5,9 a 8,5). Tendo em vista o fenômeno de imprinting, análise de metilação foi também realizada em um subgrupo de 52 pacientes com PPC pela técnica de MS-MLPA, mas não foram encontradas alterações no padrão de metilação. Em conclusão, este trabalho identificou um novo gene associado ao fenótipo de PPC. Atualmente, mutações inativadoras no MKRN3 representam a causa genética mais comum de PPC familial (33%). O MKRN3 é o primeiro gene imprintado associado a distúrbios puberais em humanos. O mecanismo preciso de ação desse gene na regulação da secreção de GnRH necessita de estudos adicionais
Most cases of central precocious puberty (CPP) in girls remain idiopathic. The hypothesis of a genetic cause has been strengthened after the discovery of some genes associated with this phenotype, particularly those involved with the kisspeptin system (KISS1 and KISS1R). However, genetic defects in KISS1 and its receptor are rare and have been identified in only a few patients with CPP.over the past years. To date, most genetic studies in CPP was based mainly on a candidate gene approach, including genes selected in animal studies, human models of patients with hypogonadotropic hypogonadism or in genome wide association studies. In the present study, we used whole exome sequencing, a more advanced method of sequencing, to identify variants associated with CPP. Thirty-six patients with the familial form of CPP (19 families) and 213 apparently sporadic cases were initially selected. The familial form was defined by the presence of more than one member affected in the family. Genomic DNA was extracted from peripheral blood leukocytes in all patients. Whole exome sequencing performed by ILLUMINA technique in 40 members of 15 families with CPP, identified inactivating mutations in a single gene, MKRN3, in five out of these families. Analysis of MKRN3 mutations performed by automatic sequencing in two additional families (four patients) identified two novel mutations. MKRN3 is an introless gene located on chromosome 15, in the Prader Willi syndrome critical region, and it is expressed only by the paternal allele due to the maternal imprinting. Following the initial findings, we searched for MKRN3 mutations in 213 patients with apparently sporadic CPP using polymerase chain reaction followed by direct enzymatic purification and automated sequencing (Sanger). Three new mutations and two previously reported, including four frameshifts and one missense variant was identified in six unrelated girls with CPP. All variants were not described in the two databases (1000 Genomes and Exome Variant Server). The familial segregation analysis performed in three out of these girls with apparently sporadic CPP and MKRN3 mutations confirmed the autosomal dominant inheritance with complete penetrance and exclusive transmission through the paternal allele, revealing familial inheritance in apparently sporadic cases. Most of these MKRN3 mutations were frameshifts or nonsense, leading to premature stop codons and truncated proteins, thus demonstrating positive genotype- phenotype correlation. The two missense mutations (p.Arg365Ser and p.Phe417Ile) identified were located within zinc finger motifs, regions predicted to be essential for the protein function. Besides that, all missense mutations were predicted to be pathogenic by in silico analysis. All patients carrying MKRN3 mutations exhibited typical clinical and hormonal features of premature activation of the reproductive axis. The median age of puberty onset was 6.0 years in girls (ranging from 3.0 to 6.5) and 8.0 years in boys (ranging from 5.9 to 8.5). In view of the imprinting phenomenon, methylation analysis was also performed in a subgroup of 52 patients with CPP by MSMLPA technique, but no methylation abnormalities were detected. In conclusion, our work has identified a new gene associated with CPP. Currently, inactivating mutations in MKRN3 represent the most common genetic cause of familial CPP (33%). MKRN3 is the first imprinted gene associated with pubertal disorders in humans. However, its precise mechanism of action in the regulation of GnRH secretion needs further studies

Le développement de l'axe gonadotrope (HHG) débute pendant la vie foetale mais ne s'achève qu'une fois la puberté terminée. De nombreux acteurs interviennent à chaque étape et le défaut de l'un d'entre eux peut mener à des pathologies de la puberté ou des troubles de la fertilité à l'âge adulte. Les facteurs génétiques ont une place centrale dans le développement de l'axe HHG et l'étude génétique des pathologies de la puberté a permis des avancées majeures dans la compréhension des mécanismes moléculaires sous-jacents, bien qu'il subsiste toujours de nombreuses inconnues. Pour mon travail de thèse, j'ai choisi d'explorer la génétique des maladies de la puberté afin de mieux comprendre les mécanismes étiopathogéniques de ces maladies complexes. Dans un premier temps, j'ai eu l'opportunité d'étudier une famille consanguine au sein de laquelle deux soeurs présentaient une absence de puberté associée à une augmentation des concentrations d'oestradiol et des gonadotrophines. Nous avons mis en évidence un variant rare à l'état homozygote dans le récepteur alpha de l'oestradiol (ERalpha). L'étude in vitro du récepteur muté a montré une diminution de son activité régulatrice sur un promoteur contenant des éléments de réponse à l'oestradiol ainsi qu'une activation paradoxale ligand-indépendante du promoteur de KISS1. L'étude de ces cas permet de mieux comprendre les conséquences des mutations perte de fonction de ERalpha ainsi que les mécanismes de régulation exercés par l'oestradiol via ERalpha. Dans un second temps, je me suis intéressée à la génétique de la puberté précoce centrale (PPC) et particulièrement au gène MKRN3 (Makorin ring finger protein 3) puisqu'il s'agit de la cause génétique la plus fréquente de la PPC. MKRN3 est un gène soumis à empreinte maternel dont la fonction protéique n'est pas connue. La détermination de la pathogénicité des variants faux-sens associés à la PPC repose presque exclusivement sur les analyses in silico. Dans cette partie de mon travail, j'ai montré que les outils usuels d'analyse in silico ne sont pas performants pour déterminer la pathogénicité des variants faux-sens rares de MKRN3. J'ai également proposé une nouvelle approche pour annoter la pathogénicité des variants basée sur l'analyse de la contrainte mutationnelle de MKRN3 et la conservation des acides aminés au sein de la famille des protéines MKRN. Les PPC avec transmission maternelle représentent la majorité des PPC familiales et ne sont pas expliquées par une mutation de MKRN3. J'ai cherché à identifier de nouveaux gènes impliqués dans la PPC de transmission maternelle, en partant de l'hypothèse qu'il pourrait exister un gène majeur selon un modèle monogénique. Pour cela, j'ai sélectionné 27 patients provenant de 18 familles chez qui l'analyse d'un panel de gènes associés à la PPC n'était pas contributive. L'analyse des variants sur les régions codantes combinée à l'analyse des variations du nombre de copies (CNV) sur l'ensemble du génome m'a permis d'identifier des gènes candidats dont la fréquence a été évaluée sur une cohorte réplicative de 48 patients par séquençage à haut débit (NGS). Cette analyse n'a pas permis d'identifier un gène majeur. Toutefois, nous avons identifié des variants perte de fonction dans deux gènes pour lesquels l'analyse de l'expression hypothalamique chez la souris a montré une diminution pendant la phase juvénile, suggérant leur implication dans le contrôle post-natal de la maturation de l'axe HHG. Cette troisième partie indique que la PPC est une maladie génétique complexe. Ce travail de thèse permet de mieux comprendre les conséquences cliniques et biologiques de la perte de fonction de ERalpha. Il confirme la complexité du contrôle génétique du développement et de la maturation de l'axe HHG. Finalement, il montre que l'annotation des variants pour les maladies de la puberté est complexe et que les analyses in-silico actuelles ne sont pas adaptées à l'étude de la PPC
The development of the gonadotropic axis (HHG) begins during fetal life but is not completed until puberty. Numerous players are involved at each stage, and a defect in any one of them can lead to pubertal pathologies or fertility disorders in adulthood. Genetic factors play a central role in the development of the HHG axis, and the genetic study of pubertal pathologies has led to major advances in our understanding of the underlying molecular mechanisms, although there are still many unknowns. For my thesis work, I chose to explore the genetics of pubertal diseases in order to better understand the etiopathogenic mechanisms of these complex disorders. First, I had the opportunity to study a consanguineous family in which two sisters showed an absence of puberty associated with increased concentrations of estradiol and gonadotropins. We identified a rare homozygous variant in the estradiol receptor alpha (ERalpha). In vitro study of the mutated receptor showed a decrease of its regulatory activity on a promoter containing Estradiol Response Elements, as well as a paradoxical ligand-independent activation of the KISS1 promoter. The study of these cases provides a better understanding of the consequences of ERalpha loss-of-function mutations and the regulatory mechanisms exerted by estradiol via ERalpha. Next, I focused on the genetics of central precocious puberty (CPP), and in particular the MKRN3 (Makorin ring finger protein 3) gene, since its mutations are the most common genetic cause of CPP. MKRN3 is a maternally imprinted gene whose protein function is unknown. Determining the pathogenicity of CPP-associated missense variants relies almost exclusively on in silico analyses. In this part of my work, I have shown that the usual in silico analysis tools do not efficiently determine the pathogenicity of rare MKRN3 missense variants. I have also proposed a new approach to annotate the pathogenicity of variants based on the analysis of MKRN3 mutational constraint and amino acid conservation within the MKRN protein family. Maternally inherited CPP accounts for the majority of familial CPP and is not explained by a mutation in MKRN3. I aimed to identify new genes involved in maternally inherited CPP, based on the hypothesis that a major gene might exist in a monogenic model. For this purpose, I selected 27 patients from 18 families in whom analysis of a panel of genes associated with CPP was non-contributory. Analysis of variants in coding regions combined with genome-wide copy number variation (CNV) analysis led to the identification of candidate genes whose frequency was assessed on a replicative cohort of 48 patients by high-throughput sequencing (NGS). This analysis failed to identify a major gene. However, we did identify loss-of-function variants in two genes for which mouse hypothalamic expression analysis showed a decrease during the juvenile phase, suggesting their involvement in the post-natal control of HHG axis maturation. This study shows that CPP is a complex genetic disease. My research provides a better understanding of the clinical and biological consequences of loss of ERalpha function. It confirms the complexity of genetic control of development and maturation of the HHG axis. Finally, it shows that the annotation of variants for pubertal diseases is complex and that current in-silico analyses are not adapted to the study of CPP

Tese (doutorado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2018.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação de Apoio à Pesquisa do Distrito Federal (FAP-DF).
CAPÍTULO I: Neste trabalho, foi estudada uma família na qual 3 irmãs apresentaram amenorréia primária por provável alteração na formação dos ductos de Müller (DMs), caracterizada por hipoplasia uterina, endométrio não responsivo a estrógenos e gestações tubárias. Através de sequenciamento exômico amplo seguido por análise genética abrangente foi identificado uma mutação em homozigose no gene Odd-skipped related 1 gene (OSR1), p.V108F. Para esclarecer os efeitos do Osr1 no desenvolvimento dos DMs, foram investigados o padrão de expressão pré-natal e pós-natal de Osr1/Osr1 nos DMs e endométrio, respectivamente, e se a deleção de Osr1 poderia afetar o desenvolvimento dos DMs, através do uso de camundongos geneticamente modificados. Foi demonstrado que o Osr1 é expresso nos DMs e nos ductos de Wolff (DWs) de embriões com 13,5 dias de gestação (E13,5). Curiosamente, os DMs não foram observados no lado esquerdo e estavam truncados rostralmente no lado direito de E13,5 Osr1 -/- nocautes. Após o nascimento, o Osr1 é expresso no útero de camundongos selvagens ao longo de todo o desenvolvimento, com expressão mais acentuada em dois períodos distintos, aos 14 dias pós natais (PND14) e PND28-PND35, que correspondem à adenogênese endometrial e o início da puberdade, respectivamente. No útero adulto, a proteína Osr1 é expressa principalmente nas células epiteliais luminais e glandulares do endométrio, como também no epitélio dos ovidutos, sendo observada menor expressão no estroma endometrial. Através de uma abordagem translacional, demonstramos que OSR1 é um novo candidato entre os fatores moleculares que modulam a formação e diferenciação de estruturas derivadas dos DMs. CAPÍTULO II: No presente estudo, foram investigados comparativamente o padrão de expressão de Mkrn3 no hipotálamo com as gônadas masculinas e femininas. Além disso, foram abordados o padrão de expressão temporo-espacial desta proteína durante o desenvolvimento sexual, e se ela é regulada nos compartimentos testiculares pelas gonadotrofinas. A quantificação por qPCR mostrou que os níveis de mRNA de Mkrn3 foram detectados em testículos e ovários de camundongos selvagens em todas as idades avaliadas, entretanto, o padrão de expressão de Mkrn3 foi dimórfico entre gônadas masculinas e femininas ao longo da vida. Curiosamente, a expressão de Mkrn3 foi maior entre PND28 e PND35 nos testículos, enquanto que nos ovários atingiu os menores níveis durante o mesmo período. Adicionalmente, a coloração de X-gal em cortes de testículos provenientes de camundongos Mkrn3-LacZ adultos mostrou que o Mkrn3 é principalmente localizado no compartimento intersticial, especificamente em células de Leydig, mas também foi detectado nos túbulos seminíferos com menor expressão. Estudos in vitro e in vivo demonstraram que o RNAm de Mkrn3 aumentou em culturas primárias de células de Leydig tratadas com hCG. Além disso, a administração aguda de agonista de GnRH em camundongos selvagens adultos aumentou a expressão de Mkrn3 nos testículos, enquanto a inibição do eixo HPG pela mesma substância administrada de forma crônica levou ao efeito oposto. Por fim, no grupo de animais que receberam injeção de hCG após a inibição do eixo HPG, foi observado aumento na expressão de Mkrn3. Em conjunto, análises de expressão durante o desenvolvimento e estudos in vitro e in vivo mostraram que o Mkrn3 é produzido nos testículos, predominantemente nas células de Leydig, e que sua expressão de RNAm aumenta após a puberdade e é responsiva à ativação do receptor LH/hCG.
CHAPTER I: We present a family in which three sisters had a Mullerian Duct (MD) anomaly characterized by uterine hypoplasia, estrogen-unresponsive endometrium, primary amenorrhea, but spontaneous tubal pregnancies. Whole Exome Sequencing followed by comprehensive genetic analysis identified a novel homozygous variant in Odd-skipped related 1 gene (OSR1), p.V108F. To clarify the effects of Osr1 on MD development, we investigated prenatal and postnatal expression patterns of Osr1/Osr1 in the MDs and endometrium, respectively, and whether Osr1 deletion affects MD development, using genetically engineered mice. We showed that Osr1 is expressed in the MDs and Wolffian ducts (WDs) of E13.5 embryos. Interestingly, MDs are absent on the left side, and rostrally truncated on the right side of E13.5 Osr1-/-knockouts. Osr1 is expressed lifelong in WT mice uterus with two distinct peaks at PND14 and PND28-PND35, which correspond to endometrial adenogenesis and puberty initiation, respectively. Osr1 is expressed mainly in endometrial luminal and glandular epithelial cells, and less in stroma, with a high expression in oviduct epithelium. This pair-rule gene plays critical roles on embryonic patterning and tissue morphogenesis. Through a translational approach, we demonstrated that OSR1 is a novel candidate among the molecular factors that modulate the formation and differentiation of MD-derived structures.CHAPTER II: In the present study, we comparatively investigated the behavior of Mkrn3 expression in the hypothalamus versus male and female gonads. We also addressed the temporo-spatial expression pattern of this protein during sexual development, and whether it is regulated in the functional testicular compartments by gonadotropins. Quantification by qPCR showed that Mkrn3 mRNA levels was detected in testes and ovaries of wild-type mice at all ages evaluated, however, the pattern of Mkrn3 expression across lifespan differed between male and female gonads. Interestingly, Mkrn3 expression was highest by PN28 to PN35 in the testes, whereas it reached the nadir at the same postnatal ages in the ovaries. Moreover, X-gal staining of testes sections from adult Mkrn3-LacZ reporter mice showed that Mkrn3 is expressed mainly in the interstitial compartment, specifically in Leydig cells, but was also mildly detected in the seminiferous tubules. In vitro and in vivo studies demonstrated that the Mkrn3 mRNA levels increased in hCG-treated Leydig cells primary cultures. Furthermore, the acute administration of LHRH agonist in adult wild-type mice increased Mkrn3 expression in testes and the inhibition of the HPG axis, by chronic administration of LHRH agonist, leads to the opposite effect. Finally, the rescue of Mkrn3 expression was observed in the group of animals that received hCG injection after completing the HPG downregulation phase. Taken together, our developmental expression analyses, in vitro and in vivo studies showed that Mkrn3 is produced in the testis, predominantly in the Leydig cells, and that its mRNA expression increases after puberty and is responsive to LH/hCG receptor activation.

Telomeres are important structural and functional components of chromosomes, serving to provide stability and enabling full replication of the chromosomes. However, a shortening of the telomeres occurs with each cell division that can be fixed by a polymerase activity provided by telomerase, preventing this loss which would otherwise eventually lead to chromosome end-to-end fusions, senescence and cell death. The telomerase activity is present in stem cells and germ line cells, but absent or barely noticeable in adult somatic cells. However, in approximately 80-90% of transformed somatic cells the telomerase activity is recovered, resulting in a "telomerase positive phenotype". This phenotype has been a prime target in cancer research, and recently a novel mechanism for regulating telomerase levels has been uncovered. Makorin 1 RING finger protein (MKRN1) was found to be an E3 ubiquitin ligase for hTERT, the rate-limiting catalytic component of telomerase, leading to the ubiqutin-mediated 26s proteasomal degradation of hTERT and reduced telomerase activity. So, MKRN1 plays a role in telomere homeostasis. In this study we looked at the expression of MKRN1 in numerous tumor cell lines (Hela, HCT116, HL60) and the normal diploid fibroblasts (WI-38). In the latter cell line, basal levels of MKRN1 were found to increase 6-fold when the cells were serum starved and arrested in G1/G0. In contrast, the cancer cell lines expressed MKRN1 at low levels or undetectable. This would indicate that MKRN1 is up-regulated in resting or G1 arrested cells.In one cell line the promyelocytic leukemia, HL-60, showed no protein levels of MKRN1. This cell line is able to be terminally differentiated upon ATRA treatment, when cells are arrested at G1. In this model system of cellular differentiation hTERT mRNA levels and telomerase activity decrease drastically and quickly. We hypothesized that the differentiation of HL-60 induced by ATRA would be accompanied by an increase in MKRN1 levels. MKRN1 mRNA and protein levels were strongly up-regulated during the ATRA-mediated differentiation of HL-60 cells. Although, a decrease in hTERT mRNA is a contributor to telomerase inhibition during cellular differentiation; our data indicate that the up-regulation of MKRN1 ensures the effective removal of residual telomerase activity by the ubiquitin-mediated degradation pathway at the proteasome.
M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular and Microbiology MS

Age at pubertal initiation is associated with the risk of many severe health outcomes later in life. “Reactivation” of the hypothalamic-pituitary-gonadal axis, signified via an increase in the amplitude and frequency of gonadotropin-releasing hormone, (GnRH) marks the commencement of puberty. For decades, physicians and scientists have attempted to unravel the mysterious mechanisms underlying the onset of this transitional period of reproductive maturation. In 2013, loss-of-function mutations in maternally imprinted Makorin RING Finger 3 (MKRN3) were determined to be the one of the causes of central precocious puberty (CPP), establishing MKRN3 as the first inhibitory gene with mutations in humans indicated in the upstream regulation of GnRH. MKRN3 accomplishes this suppression through its function as an E3 ligase, marking components involved in the transcription of kisspeptin and neurokinin B, two stimulatory regulators of GnRH, for degradation by the proteasome. MKRN3 remains the most common genetic cause of CPP to date. As a maternally imprinted gene, loss-of-function mutations in MKRN3 have only been shown to produce a phenotype in patients when inherited paternally. However, the mechanisms conferring monoallelic expression of MKRN3 are not well understood – nor are the downstream implications of MKRN3 imprinting abnormalities on the development, or even simply, association, with pubertal disorders. It has been determined that differential methylation – a common epigenetic modification involved in the demarcation of imprinted genes – plays a role in the imprinting mechanism of MKRN3. The purpose of this study is to characterize the differentially methylated region (DMR) and to expand understanding of how this region may serve to mark the maternal allele for repression. This will set the foundation for subsequent studies into the potential contribution of methylation defects in the MKRN3 DMR toward the onset of pubertal disorders. Two potential regulatory sites decorated with CpG islands were identified: the proximal-promoter and a potential enhancer. In classical imprinting studies, differential methylation of the proximal-promoter has been shown to mark alleles on a parent-specific basis for transcriptional inhibition – subsequently silencing expression dependent on parental origin. Additionally, many studies describe a similar process with an upstream or downstream enhancer region being the site influencing transcriptional activity of the alleles on a parent-of-origin basis; preliminary studies indicate a region 3.7 kb upstream of the MKRN3 transcription start site which may act as an enhancer for MKRN3. In this study, DNA was extracted from whole, peripheral blood of individuals with normal pubertal development, followed by bisulfite conversion and PCR amplification, with clean-up and submission for next generation sequencing. Sequencing analysis allowed for the quantification of the methylation profile of each sample at its respective regulatory site. The proximal-promoter was found to be almost entirely methylated while the enhancer exhibited differential methylation, but with notable variability between samples. We identified this enhancer region as the presumptive DMR of MKRN3. Additional research is required to confirm the status of the proposed enhancer as the DMR regulating the paternal-specific expression of MKRN3.

BACKGROUND: MKRN3 is a gene recently identified to encode the first known inhibitor of puberty initiation. MKRN3 mutations have been identified in children diagnosed with familial central precocious puberty. MKRN3 is a maternally imprinted gene; only the father’s allele is expressed by the child. Family studies and patterns of inheritance affirm that mutant alleles only result in CPP when the mutation was inherited from the father. Although mutations in MKRN3 were found to have implications in development of central precocious puberty, its mechanism of action remains a mystery. Previous studies in our laboratory have focused on MKRN3’s function as an E3 ubiquitin ligase and thus sought to investigate its potential targets in the cell. Because kisspeptin is the most potent known activator of GnRH neurons, it seemed to be the most likely candidate. Despite efforts to identify an association between kisspeptin levels and MKRN3 expression, little headway has been made. OBJECTIVE: Beyond the most common known function of E3 ubiquitin ligases in protein degradation, increasing evidence suggests that E3 ubiquitin ligases contribute to aiding human brain development throughout childhood and into adolescence. This proposed action of E3 ubiquitin ligases led us to propose a role for MKRN3 as a potential regulator of GnRH neuronal plasticity and maturation. I hypothesized that MKRN3 plays a role in delaying GnRH neuronal maturation and neuronal plasticity until puberty onset. Thus, MKRN3 deficiency would result in premature GnRH neuron maturation. METHODS: A mouse model of Mkrn3 knockout mice (Mkrn3 +/p-) was compared to wildtype mice (Mkrn3+/+). Using DAB IHC, GnRH neurons in the rostral pre-optic area (rPOA) area were labeled and morphologically analyzed. Additionally, Golgi staining of neurons in the arcuate nucleus was done to visualize details of neuronal synapses. Neurons were visualized under 40X and 100X magnification. To visualize spines, confocal images of dendrites from Mkrn3+/p- and Mkrn3+/+ mice were captured. Images were visualized and analyzed using both ImageJ and NeuronStudio software (for spine analysis only). RESULTS: Initial morphological analysis of GnRH neurons failed to show any significant effect of the Mkrn3 genotype on dendrite arrangement, varying among complex, bipolar, and unipolar. Similarly, Golgi staining analysis revealed that the Mkrn3 genotype did not have a measurable effect on dendritic spine type percentages. Mkrn3+/p- and Mkrn3+/+ mice showed no apparent difference in the percentage of stubby, thin, and mushroom shaped spines. However, Mkrn3 deficiency did show an effect on spine density, with neurons from the arcuate nucleus of the hypothalamus of Mkrn3+/p- mice having an increased total number of dendritic spines compared to neurons from wildtype mice. CONCLUSIONS: Mkrn3 does not appear to have an effect on dendrite architecture of GnRH neurons nor change in spine type of arcuate nucleus neurons. However, the Mkrn3 genotype does seem to affect neuronal spine density, at least in the population of neurons in the arcuate nucleus. Future research is needed to conclusively determine the mechanism of action of MKRN3 and its specific role in central precocious puberty.

碩士
國立陽明大學
解剖學及細胞生物學研究所
102
The ether-à-go-go (Eag) potassium channel belongs to the EAG family of voltage-gated K+ (Kv) channels. Two Eag isoforms have been identified in rat, Eag1 (rEag1) and Eag2. In mammals, the expression of Eag1 potassium channels is neuron-specific and is widely distributed over various brain regions. Despite of their abundant expression in the nervous system, the neurophysiological role of Eag1 potassium channels remains obscure. To better understand the physiological roles of Eag1 channels, we heve applied the yeast two-hybrid screening system to identify rEag1-interacting proteins from a rat brain cDNA library. One of the clones we identified was Mkrn1, an E3 ubiquitin ligase that targets the hTERT (human telomerase reverse transcriptase) for proteasome processing, decreasing telomere activity and subsequently telomere length. GST pull-down assay and co-immunoprecipitation assay were performed to confirm the interaction between rEag1 and Mkrn1. To investigate whether Mkrn1 functions as an E3 ubiquitin ligase and mediates ubiquitination and degradation of rEag1, cells were treated with cycloheximide (CHX) to inhibit protein synthesis and the degree of rEag1 ubiquitination was exmined. The data demonstrated that Mkrn1 overexpression increased the ubiquitination and decreased the half-life of rEag1 proteins. When co-expressed Mkrn1 and rEag1 in HEK293T cells, in addition to migrating as a doublet, a third low molecular-weight band was detected by Western blotting analysis with anti-rEag1 antibody. Deglycosylation treatment showed that the third band had the molecular weight similar to the deglycosylated form of rEag1, suggesting that Mkrn1 might cause the deglycosylation of rEag1 during the ubiquitination-proteasomal degradation. In addition to the heterologous expression system, we have also performed the immunofluorescence and subcellular fractionation analysis to demonstrate the co-localization of rEag1 and Mkrn1-long in native hippocampal neurons and forebrain lysates. In this study, we have combined morphological and biochemical methods to investigate the physiological significance of the interaction between Mkrn1-long and rEag1. Together, our data indicates that Mkrn1-long interacts with rEag1 to induce the ubiquitin-mediated degradation of rEag1 K+ channel.