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INTRODUCTION: Pubertal growth is an important time during development for bone accrual and attainment of peak bone mass. Suboptimal bone gain has been observed in females with reproductive abnormalities such as primary and secondary amenorrhea and these conditions are very prevalent in female athletes. Amenorrhea is associated with decreased estradiol levels. Previous research has shown that in prepubertal animals a low estrogen environment significantly decreased mechanical strength, but there was no significant loss in bone area and actually an increase in moment of inertia. The decrease in mechanical properties may be related to the microstructure of the bone. Two types of bone are involved in growth-- woven bone, which is added for structural support in the short term, and lamellar bone , which is highly organized and has a greater contribution to overall strength. We will test the hypotheses that suppressed estradiol will result in bones with no change in cortical area and decreased strength properties but will have a larger composition of non lamellar bone as opposed to lamellar bone. PURPOSE: The goal of this study was to determine the relative amounts of woven and lamellar tissue in a bone and the relationship with the bone's mechanical strength in two models of low estrogen-- pre- and post-pubertal onset. METHODS: Fifty-Five female Sprague-Dawley rats were randomly assigned into four groups: a control group (n=14) and three experimental groups injected with gonadotropin releasing-hormone antagonist (GnRH-a)-- the Dose 1 group was injected with 1.25 mg/kg/dose daily (n=14), the Dose 2 was injected with 2.5 mg/kg/dose daily (n=14), and the Dose 3 group was injected with 5.0 mg/kg/dose, 5 days per week (n=13). All groups were sacrificed at Day 49. Additionally, twenty-nine Sprague Dawley rats were randomly assigned into three groups. The baseline day 65 group (BL 65) was sacrificed on day 65 (n=9). There was an aged match control group that was sacrificed on day 90 (n=12). Finally, there was an AMEN experiment group injected with 2.5 mg/kg/dose daily that was sacrificed on day 90 (n=9). All experimental groups for both protocols received injections of gonadotropin releasing hormone antagonists (GnRH-a) (Zentaris GmbH) intraperitoneally. Left femora were mechanically tested under 3-point bending. The right femora were dehydrated, embedded in polymethylmethacrylate, cut and ground to 100 µm thickness. Bones were analyzed under polarized light using Stereo Investigator Software (MBF Bioscience, VT). The proportion of the cortex with primary lamellar vs. non-lamellar/other primary tissue type was measured and expressed as percent of the total cortical bone area. Outcome measures included lamellar endocortical area, lamellar periosteal area, cortical area, endocortical area, % lamellar area and % non-lamellar area. RESULTS: There was a significant decrease (p<.05) in the distribution of lamellar versus non-lamellar cortical tissue type in the experimental group in the model of delayed puberty. Additionally, the pre-pubertal bones had a lower percentage of lamellar periosteal and endocortical area. The post-pubertal group showed no significant differences between the control and experimental group in any of the outcome measures. CONCLUSION: There were significant differences in relative bone distribution throughout the femoral cortex. Relative decreases in lamellar tissue distribution, especially on the periosteal surface, will result in decreased mechanical strength due to increased percentage of woven bone in pre-pubertal models. / Kinesiology / Accompanied by one .pdf file: Lamellar/Woven Database.

Osteoporosis which is frequently referred to as a pediatric disease with geriatric consequences (Golden, 2000) can result from a lack of optimal bone accrual during the development (NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis, and Therapy, 2001). Pubertal timing is a key factor that contributes to optimal bone accrual and strength (Bonjour et al., 1994; 21 Warren et al., 2002). Bone mass doubles during the onset of puberty and young adulthood (Katzman et al., 1991) with more than 90% of peak bone mass being accrued at the end of second decade of life (Schneider & Wade, 2000). The rate of periosteal expansion is elevated during the pubertal period (Specker et al., 1987; Bradney et al., 2000) and this expansion parallels longitudinal growth (Parfitt, 1994). Irrespective of other changes, periosteal expansion lowers fracture risk by improving the strength of long bones by increasing the moment of inertia (Orwoll, 2003). Therefore, a delay in puberty may actually increase the time available for periosteal development and positively affect bone strength. Previous animal studies have shown decreases in strength, endocortical bone formation and increases in periosteal bone formation with delayed puberty. Clinical studies report negative effects of delayed puberty on bone mass accrual suggesting that delayed puberty is a multifactorial problem affecting bone strength development. The purpose of this study was to determine the effect of delayed puberty on mechanical strength and endocortical bone marrow cells in two models: female rats treated with gonadatropin releasing hormone antagonists (GnRH-a) and energy restriction (30%). Thirty-two female Sprague Dawley rats (21 to 22 days-of-age) were received from (Charles Rivers Laboratories, Wilmington, MA, USA) and housed individually at the Temple University Central Animal Facility (Temple University Weiss Hall). Animals were randomly assigned to one of three groups; control (n=10), GnRH-a (n=10) and energy restriction (ER) (n=12). The GnRH-a group was injected with gonadotropin releasing antagonist injections (GnRH-a) (Antide, Bachem, Torrance, Ca. USA) at a dose of 2.5 mg/kg/BW. The ER group received a 30% energy restricted diet (0pen Source diet (D07100606)(Research Diets, New Brunswick, NJ). All animals were sacrificed on Day 51. One way analysis of variance testing (ANOVA) with a significance level of 0.05 was used to assess group differences. Following the two protocols the uterine weight in the GnRH-a group was 80.6% lower than control; no change in the ER group. Ovarian weight was significantly lower in the GnRH-a group (83.3%) and in the ER group (33.3%) as compared to controls. A 22.7% lower muscle weight was found in the ER group but was equal to control and GnRH-a when normalized by body weight (BW). The retro-peritoneal fat pad weight was significantly decreased by 64.95% in the ER group as compared to controls. Energy restriction did not result in any deficit in bone strength when normalized by body weight however the GnRH-a group had a 26.2% lower bone strength compared to control. Histomorphometric changes were not significantly different between groups, but the ratio for periosteal versus endocortical bone formation rates for the control group was 1.38, GnRH-a was significantly higher with a ratio of 5.54 and for ER was 3.02 indicating that periosteal BFR are almost twice endocortical BFR in the experimental groups. There was a significant decrease in the trabecular percent bone volume (BV/TV) of the lumbar vertebra in the GnRH-a group (20.2%) compared to control. However BV/TV was significantly higher in the ER (18.4%) compared to the control group. Proliferation was suppressed to 59.6% of control in the GnRH-a group but only 85.5% of control in the ER group. The alkaline phosphatase activity was 31.2% lower in the GnRH-a group and 63.9% lower in the ER group. The relative quantification (RQ) of RUNX2 gene expression was lowest in control followed by GnRH-a and highest in ER group although no statistical significance was observed between any groups. Thus our data infers that 30% energy restriction does not negatively impact bone health. Thirty percent food restriction with no deficits in micronutrients or hormone suppression may just suppress growth as indicated by the maintenance of bone strength per body weight and equivalent muscle mass per body weight in the ER group compared to control. The GnRH-a injections resulted in decreased bone strength and trabecular bone volume. Female Athlete Triad or Anorexia Nervosa are the two clinical conditions hypothesized to result from a combination of ER and estrogen deficient environment. Studies replacing estrogen in hypothalamic amenorrhea or IGF-1 in anorexia alone have failed to improve bone mineral density (BMD), but a combination of IGF-1 and estrogen has been successful in improving BMD. This suggests that estrogen dependant and independent mechanisms work in combination to protect bone. Our study investigated both mechanisms separately and indicates that ER at 30% may be protective for bone health. Since estrogen deficiency may be the extreme end of the spectrum affecting trabecular bone, treatment therapies may have to be based on age, magnitude and severity of energy restriction and presence or absence of menstrual status. / Kinesiology

The purpose of this investigation was to determine the effects of post pubertal caloric restriction on bone architecture, strength, and medullary adipose quantity. A randomized control comparison design was utilized and the study was conducted in a laboratory setting. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Temple University (protocol number 3396). Female Sprague Dawley rats (23days-of-age, n=120) were randomly assigned into seven groups, baseline (BL) (n=18), control (C) (n=17), caloric restriction (FR) (n=17), control recovery (RC) (n=17), caloric restriction recovery (RFR) (n=17), control ovariectomy (COVX) (n=17) and food restricted ovariectomy (FROVX) (n=17). On day 65, a 6 week 30% caloric restriction protocol was administered. Following food restriction, a subset of the control and food restricted groups were sacrificed (n=34) and the remaining animals (n=68) control recovery (RC) and food restricted recovery (RFR) groups had a 10 week recovery with ad lib food. Recovery groups, RC and RFR: were sacrificed after the 10 week recovery period at 183 days of age (n=34). The remaining animals were ovariectomized (OVX) and grouped into control ovariectomy (COVX) and food restricted ovariectomy (FROVX). Six weeks post OVX the animals were sacrificed at 270 days of age. After sacrifice blood was taken by cardiac puncture, bones were harvested, cleaned of soft tissue, fixed and prepared for analysis. Anthropometric measurements were taken including retroperitineal and gonadal fat pad weights as well as adrenal glands, ovaries, uteri, and tricep surae muscle group weights. Main Outcome Measures: The outcome variables for this study were bone mechanical competence, trabecular and cortical bone mass and architecture, marrow adipocyte number as well as serum markers of bone formation and resorption. Insulin - like growth factor - 1 (IGF-1) and C- terminal telopeptide (CTX) was measured to determine bone formation and resorption. Statistical Analysis: One-way Analysis of Variance (ANOVA) was performed to determine differences between all groups. Tukey's honestly significant difference (HSD) post hoc analysis was conducted to determine differences between groups. Student's t - tests were used to detect differences between age groups (acute, recovery, post-OVX) A p value was set at less than or equal to 0.05 for all statistical tests. All statistical analysis was performed using (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA). Variables were normalized with a linear regression-based correction using body weight. All variables with an R2 level greater than 0 were normalized to avoid choosing an arbitrary R2 value as a cut-off for normalization. Results: Body weight was 18% lower than control animals following caloric restriction. Weight loss was due to fat mass predominately; muscle mass was maintained relative to body weight. Bone length and growth rates were diminished however no differences were found following refeeding. No differences were found in bone strength at any time point. However relative to body weight peak moment and stiffness were significantly higher following caloric restriction. Cortical bones mass and cross sectional moment of inertia were enhanced in the femoral diaphysis with bone mass greater post OVX in the calorically restricted group (FR-OVX). No significant differences were found in ash percent in the femur was found between any groups at any time point however vertebral bone mineral density in acute FR and post OVX time points in FROVX was significantly greater indicating an enhanced bone quality in the restricted. No change in trabecular quantity or quality were observed in the distal femur between groups however vertebral trabecular architecture was enhanced in number and thickness in acute FR and post OVX time points in FROVX. No significant difference in number of marrow adipocytes were found at any time point. Serum CTX decreased significantly in acute in FR and increased at recovery in RFR and post OVX in FROVX. Serum IGF - 1 decreased in the acute FR with IGF - 1 significantly greater after recovery in RFR. Conclusions: Evidence was found to suggest that moderate caloric restriction (nutrient replete) post puberty was positive for bone. Bone quantity was increased with relative cortical area and bone area relative to body weight increased in the FR group. Significant increases in FROVX bone quantity post OVX suggests that bone mass gains during caloric restriction attenuated cortical bone loss at maturity post OVX. Bone quality increases in cross sectional moment of inertia relative to body weight may have accounted for the transient increase in FR bone strength in the femur. Decreases in acute CTX and IGF- 1 levels indicates that bone formation and resorption were decreased during development that may have been the mechanism for bone loss attenuated post OVX in calorically restricted. Growth rate slowing during caloric restriction may have decreased the rate of formation and resorption during a crucial time of peak bone mass accrual and bone modeling. This decrease in one modeling may have been mechanism that preserved bone quantity during acute caloric restriction. Increases in femur quality in polar moment of inertia coupled with a decrease in bone length changed the shape of the bone making it more robust. A shorter bone with a thicker cortex with no change in mineral content may have been the mechanism in the transient increase in bone strength in the femur. Quality changes in mineral density in vertebrae acting as a mineral storage back up as a last resort if quantity and quality changes were not sufficient in maintain bone strength. Moderate caloric restriction transiently increased strength, by increasing bone mass relative to body, altering bone geometry and increased vertebral mineral density. / Kinesiology

Yes / The skeletal remains of infants and children are a poignant reminder of the perilous nature of childhood in the past, yet they offer valuable insight into the life histories of individuals and into the health of populations. Many osteoarchaeological and bioarchaeological analyses are dependent on two vital pieces of information: the age-at-death and sex of the individual(s) under study. This chapter will outline how age-at-death and sex can be estimated from the skeletal remains and dental development of non-adults, and how these are easier or more difficult to determine than for adults, and will discuss the complexities and controversies surrounding different methods.

Puberty rites are practised in many countries including South Africa. In South Africa the puberty rites have different names and different practices. This study focused on vukhomba among the Manchangana/Vatsonga. Vukhomba is conducted exclusively for girls who have reached menarche. The purpose of this study was to explore the possibility of utilising vukhomba for the improvement of the health status of women. The study design is a qualitative, exploratory, descriptive contextual research study conducted in the Northern region of the Northern Province among Vatsonga\Manchangana in four selected areas. The ethnographic strategy was used to gain access to the vukhomba to view and describe the rite from an emic perspective. The sample included all girls who were initiates during January 1998 and December 1999 in the four selected areas, as well as Vadzabi, varileri, initiated girls, initiated women and vukhomba elders who attended the initaitions. The techniques for data collection included participant observation, semi-structured interviews, focus group discussions, key informant interviews and feedback workshops. The findings indicate that vukhomba is conducted during the school holidays in order to cater for girls who are still attending school. The sexuality education in this rite is mainly about encouraging initiates to maintain their virginity for their future husbands. Vukhomba therefore teaches girls attending the initiation the facts of life. It was however, found that girls often attend the initiation for material gain and respect for elders. The content of sexuality education information given to girls during the rite is inadequate. Initiated women and girls wanted to gain more knowledge about their bodies, their health, menstruation, child bearing and pregnancy, contraceptives and pregnancy. After reviewing the findings of the research an intervention programme was developed and discussed with the initiated women and initiated girls. Vukhomba elders accepted the intervention programme, however, certain topics were not approved e.g. contraception. The intervention programme is expected to form part of the initiation programme in the future. Initiated community members will be trained to facilitate the activities of this programme. / Health Studies / D.Litt. et Phil.

O complexo de sinalização kisspeptina-GPR54 é um regulador chave para ativação dos neurônios de GnRH e do eixo reprodutivo. Mutações inativadoras no GPR54 foram identificadas em pacientes com hipogonadismo hipogonadotrófico normósmico isolado (HHIn). A partir desse achado, hipotetizamos que mutações ativadoras no GPR54 resultariam na liberação prematura de GnRH e, conseqüentemente, no aparecimento de puberdade precoce, dependente de gonadotrofinas (PPDG). No presente estudo, investigamos a presença de mutações ativadoras e/ou polimorfismos em pacientes com PPDG, assim como a presença de mutações inativadoras e/ou polimorfismos em pacientes HHIn ou retardo constitucional do crescimento e desenvolvimento puberal (RCCP). Cento e catorze pacientes com distúrbios puberais centrais idiopáticos foram selecionados, sendo 53 com PPDG, 33 com HHIn e 28 com RCCP. Cento e cinqüenta controles brasileiros que relatavam desenvolvimento puberal normal foram estudados. A região codificadora do GPR54 de todos os pacientes foi amplificada utilizando-se oligonucleotídeos intrônicos específicos, seguida de purificação enzimática e seqüenciamento automático. No grupo de puberdade precoce, identificamos uma nova variante em heterozigose no exon 5 do GPR54, que se caracterizou pela troca do aminoácido arginina por prolina na posição 386 (R386P) do receptor. Esta substituição foi encontrada em uma menina adotada com PPDG e estava ausente nos controles normais. Estudos in vitro demonstraram que as quantidades de fosfatidil-inositol (IP) e o grau de fosforilação da quinase regulada por sinal extracelular (pERK) em condições basais não foram significativamente diferentes entre as células transfectadas com o receptor selvagem ou com o receptor contendo a mutação R386P, indicando que não havia ativação constitutiva do receptor. No entanto, estudos por tempos mais prolongados demonstraram que a quantidade de IP e o grau de pERK permaneceram significativamente mais altos nas células transfectadas com o receptor mutante quando comparadas ao selvagem, indicando ativação da sinalização intracelular, porém por um mecanismo não-constitutivo. No grupo de hipogonadismo, duas novas variantes foram identificadas em três pacientes. Uma mutação do tipo inserção/deleção (indel) em homozigoze no sítio aceptor de splicing no intron 2 (IVS2-4_-2delGCAinsACCGGCT) do GPR54 foi identificada em dois irmãos com HHIn. Uma troca em heterozigose, E252Q, foi identificada em um paciente com HHIn esporádico. As duas alterações estavam ausentes no grupo controle. Polimorfismos foram encontrados nos pacientes com RCCP. Em conclusão, descrevemos a primeira mutação ativadora do GPR54 associada ao fenótipo de PPDG. Descrevemos uma nova mutação inativadora em sítio de splicing em pacientes com HHIn, entretanto mutações inativadoras do GPR54 são uma causa rara de HHIn. / The kisspeptin-GPR54 signaling complex is a gatekeeper of pubertal activation of GnRH neurons and of the reproductive axis. Inactivating mutations in the GPR54 receptor were identified in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH). Based on this observation, we hypothesized that gain-of-function mutations of the human GPR54 receptor might be associated with premature activation of GnRH release, leading to gonadotropin-dependent precocious puberty (GDPP). In the present study, we investigated the presence of GPR54 activating mutations or polymorphisms in patients with GDPP and inactivating mutations or polymorphisms in patients with nIHH or constitucional delay of puberty (CDP). A hundred fourteen patients were selected; 53 with GDPP, 33 with nIHH and 28 with CDP. A hundred and fifty Brazilian controls who reported normal pubertal development were also studied. The entire coding region of GPR54 of all patients was amplified using specific intronic oligonucleotides followed by enzymatic purification and automated sequencing. We have identified a novel variant in heterozygous state in exon 5 of GPR54, R386P, in an adopted girl with GDPP. This substitution was absent in all controls. Basal inositol phosphate (IP) and phosphorilated extracellular signalregulated kinase (pERK) levels in cells transfected with WT or R386P GPR54 were not significantly different indicating that there was not a constitutive activation of the receptor. However, studies performed in more prolonged times demonstrated that the IP and the pERK levels were significantly higher in cells transfected with the mutant receptor when compared to the wild type, indicating that the signaling pathway was still activated although by a non-constitutive mechanism. In the nIHH cohort, we have identified two novel variants in three patients. The first variant was an insertion/deletion (indel) in homozygous state within the constitutive acceptor splice site of intron 2 of GPR54 (IVS2-4_-2delGCAinsACCGGCT) identified in two male siblings with nIHH. The second variant was the change E252Q in heterozygous state in a patient with sporadic nIHH. Both alterations were absent in the control population. We have found only polymorphisms in patients with CDP. In conclusion, we have described the first activating mutation in GPR54 associated with the GDPP phenotype. We have also described a novel splice site inactivating mutation in patients with nIHH however, inactivating mutations of GPR54 represent a rare cause of nIHH.

Avanços recentes na etiologia da puberdade precoce foram obtidos a partir da análise do genoma por sequenciamento global. Mutações inativadoras do gene MKRN3 representam uma causa importante de puberdade precoce central (PPC) familial (33-46% dos casos). O objetivo do estudo foi a análise do DNA genômico de pacientes com PPC de origem familial ou esporádica sem mutações deletérias no gene MKRN3. Foram selecionados 68 indivíduos com PPC (37 com a forma familial e 31, aparentemente, esporádicos). O DNA genômico foi extraído do sangue periférico ou da saliva dos pacientes com PPC. A técnica de sequenciamento genômico em larga escala (ILLUMNA -Clonal Single Molecule Array Technology - CSMA) foi usada na busca de novos genes implicados com o desenvolvimento puberal prematuro em seis indivíduos, sendo três afetados e três não afetados, pertencentes a uma grande família brasileira com PPC (Família 1). Mutações em um gene candidato foram pesquisadas em 64 pacientes por sequenciamento automático direto (método de Sanger). Em um subgrupo de pacientes, foi realizada a técnica de MLPA com sondas customizadas na busca de deleções. Por sequenciamento genômico global, foi identificado um novo complexo rearranjo no gene DLK1, caracterizado por uma deleção de, aproximadamente, 14.000 pb na região 5\' não traduzida (5\'UTR), englobando o início do exon 1, associada a uma duplicação de uma região do intron 3 de 269 pb. O gene DLK1 está localizado no braço longo do cromossomo 14 (14q32.2) e sofre imprinting materno. Este lócus está associado à síndrome de Temple, uma doença complexa com múltiplas manifestações, incluindo puberdade precoce central em até 90% dos casos. Para investigar o efeito dessa deleção genômica, as concentrações séricas da proteína DLK1 pelo método ELISA foram medidas nas pacientes afetadas da Família 1. Valores indetectáveis de DLK1 foram encontrados nestas pacientes. O fenótipo das pacientes afetadas da Família 1 caracterizou-se por uma PPC típica, sem sinais sindrômicos (excluída a síndrome de Temple). Posteriormente, por meio do sequenciamento direto, duas novas mutações inativadoras no gene DLK1 foram identificadas (p.Val272Cysfs*14 e p.Pro160Leufs*50) em duas famílias (Famílias 2 e 3) com PPC ou história de menarca precoce. O estudo de segregação nas Famílias 1 e 2 confirmou o padrão de herança autossômico dominante com penetrância completa e transmissão exclusiva pelo alelo paterno. A média de idade de início da puberdade nas pacientes afetadas do sexo feminino foi de 5,4 anos. A técnica de MLPA com sondas customizadas para o gene DLK1 não encontrou outras deleções no subgrupo estudado. Em conclusão, foram identificadas três mutações inativadoras no gene DLK1 associadas à PPC familial de origem paterna. O DLK1 é o segundo gene imprintado associado a distúrbios puberais em humanos. Este achado sugere um papel dos genes imprintados no controle da puberdade. O mecanismo pelo qual esse gene afeta a puberdade ainda é desconhecido / Recent advances in the etiology of precocious puberty were obtained from the whole-genome sequencing analysis. Inactivating mutations of the MKRN3 gene represent a major cause of familial central precocious puberty (CPP) (33%- 46% of the cases). The objective of the study was to analyze the genomic DNA of patients with familial or sporadic CPP without deleterious mutations in the MKRN3 gene. Sixty-eight individuals with CPP (37 with familial form and 31 apparently sporadic cases) were selected. The genomic DNA was extracted from the peripheral blood or saliva of patients with CPP. We used the whole-genomic sequencing technique (ILLUMNA - Clonal Single Molecule Array Technology - CSMA) searching for a new candidate genes implicated in premature pubertal development in 6 individuals, 3 affected and 3 non-affected, belonging to a large Brazilian family with CPP (Family 1). Mutations in one candidate gene were investigated in 64 patients through automatic sequencing (Sanger\'s method). In a subgroup of patients, MLPA using synthetic MLPA probes was performed to search for deletions. A new complex rearrangement in the DLK1 gene characterized by a deletion of approximately 14.000pb in the 5\' untranslated (5\'UTR), encompassing the start of exon 1, associated with a duplication of a region of intron 3 of 269 bp was identified by whole-genomic sequencing. The DLK1 gene is located on the long arm of chromosome 14 (14q32.2) and it is maternally imprinted gene. This locus is associated with Temple syndrome, a complex disorder with multiple alterations, including central precocious puberty in up to 90% of cases. To investigate the effect of this genomic deletion, a serum measurement of DKL1 protein using ELISA method was performed in the affected patients from Family 1. Undetectable serum DLK1 levels were found in these patients. The phenotype of affected patients from Family 1 was characterized by a typical CPP, without syndromic signs (excluding Temple syndrome). Posteriorly, two new inactivating mutations in the gene DLK1 were identified (p.Val272Cysfs*14 and p.Pro160Leufs*50) through direct sequencing in two families (Families 2 and 3) with CPP or precocious menarche history. The segregation studies in Families 1 and 2 confirmed the pattern of dominant autosomal inheritance with complete penetrance and exclusive transmission by the paternal allele. The average age of puberty onset in the affected female patients was 5.4 years. The MLPA technique with synthetic MLPA probes for the DLK1 gene did not find other deletions in the studied subgroup. In conclusion, we identified 3 paternally inherited inactivating mutations in the DLK1 gene associated with familial CPP. The DLK1 is the second imprinted gene associated with pubertal disorders in humans. This finding suggests a role of the imprinted genes in puberty control. The mechanism through which this gene affects puberty is still unknown

Mutações inativadoras nos genes TAC3 e TACR3, os quais codificam a neurocinina B (NKB) e o seu receptor NK3R, respectivamente, foram descritas em pacientes com hipogonadismo hipogonadotrófico isolado (HHI) normósmico. A partir desse achado, hipotetizamos que mutações ativadoras na NKB e/ou NK3R resultariam na secreção prematura de GnRH e, consequentemente, no desenvolvimento de puberdade precoce dependente de gonadotrofinas (PPDG). Nesse estudo, investigamos a presença de mutações ativadoras e/ou polimorfismos nos genes TAC3 e TACR3 em pacientes com PPDG, bem como mutações inativadoras e/ou polimorfismos nesses genes em pacientes com retardo constitucional do crescimento e desenvolvimento (RCCD), e HHI normósmico. Duzentos e trinta e sete pacientes com distúrbios puberais centrais idiopáticos foram selecionados, sendo 114 com PPDG, 50 com RCCD, e 73 com HHI normósmico. Um grupo de 150 indivíduos que apresentaram desenvolvimento puberal normal foi utilizado como controle. As regiões codificadoras dos genes TAC3 e TACR3 foram amplificadas pela reação em cadeia da polimerase, seguido de purificação enzimática e seqüenciamento automático direto. Análises in silico e in vitro foram realizadas. Um nova variante foi identificada no gene TAC3, p.A63P, em uma paciente do sexo feminino com PPDG, a qual desenvolveu puberdade aos sete anos de idade. Essa variante (p.A63P) está localizada na proneurocinina B, e análises in silico sugeriram que ela não altera sítios constitutivos de splicing e é benigna para a estrutura da proteína. A análise de segregação familiar mostrou que a mãe da paciente, a qual apresentou um desenvolvimento puberal normal, também apresentava a alteração p.A63P em heterozigose, sugerindo que essa variante não desempenha um papel direto no fenótipo de PPDG. Uma nova variante em heterozigose no gene TACR3, p.A449S, foi identificada em uma paciente do sexo feminino com RCCD, que teve início puberal aos treze anos de idade. A análise do grau de conservação da alanina na posição 449 mostrou que esse aminoácido não é conservado entre as diferentes espécies, e análises in silico sugeriram que essa variante não altera os sítios constitutivos de splicing, e é benigna para a estrutura do NK3R. Três novas variantes no NK3R foram identificadas, p.G18D, p.L58L (c.172C>T) e p.W275*, em três pacientes do sexo masculino não relacionados com HHI normósmico. As variantes p.G18D e p.L58L foram identificadas em heterozigose, enquanto que a variante p.W275* foi identificada em heterozigose associada a variante silenciosa p.L58L (c.172C>T), e em homozigose em outro paciente. Análises in silico sugeriram que a variante p.G18D poderia afetar a funcionalidade do NK3R. Estudos in vitro dessa nova variante foram realizados, e mostraram que a mesma não altera a função do NK3R, visto que o aumento na produção de fosfatidil inositol não diferiu significativamente entre o receptor mutado e selvagem. Todas as novas variantes descritas nos genes TAC3 e TACR3 não foram identificadas em 300 alelos controles. Em conclusão, nosso trabalho identificou novas variantes nos genes TAC3 e TACR3 em pacientes brasileiros com distúrbios puberais centrais idiopáticos, e confirmou o envolvimento do complexo NKB/NK3R na etiologia do HHI normósmico / Inactivating mutations of the TAC3 and TACR3 genes, which encode the neurokinin B (NKB) and its receptor, NK3R, respectively, were described in patients with normosmic isolated hypogonadotropic hypogonadism (IHH). Based on these observations, we hypothesized that gain-of-function mutations in the NKB and/or NK3R might be associated with premature activation of GnRH release, leading to gonadotropin-dependent precocious puberty (GDPP). In this study, we investigated the presence of activating mutations and/or polymorphisms in the TAC3 and TACR3 genes in patients with GDPP, and inactivating mutations and/or polymorphisms in these genes in patients with constitutional delay of growth and puberty (CDGP) and normosmic IHH. It was selected 237 patients with idiopathic central pubertal disorders: 114 with GDPP, 50 with CDGP, and 73 with normosmic IHH. Indeed, a group 150 individuals who had puberty at adequate age was used as controls. The coding regions of TAC3 and TACR3 genes were amplified by polymerase chain reaction followed by enzymatic purification and direct automatic sequencing. In silico and in vitro analyses were performed. A new heterozygous variant in the TAC3 gene, p.A63P, was identified in a Brazilian girl with GDPP who had puberty onset at seven years of age. The p.A63P variant was located in the proneurokinin B and in silico analysis suggested that this variant does not alter constitutive splice sites, and it was benign to the protein. The segregation analysis revealed that her mother was heterozygous for the p.A63P variant (who had a normal pubertal development), suggesting that this variant does not play a role in the GDPP phenotype. It was identified a new heterozygous variant, p.A449S, in the TACR3 gene in a Brazilian girl with CDGP, who had puberty onset at thirteen years of age. Conservation degree analysis of alanine at position 449 showed that this amino acid is not a conserved residue among different species. In silico analyses suggested that this new variant does not alter splice sites or affects the structure of NK3R. Indeed, it was identified three new distinct variants in the TACR3 gene, p.G18D, p.L58L (c.172C>T) and p.W275*, in three unrelated males with normosmic IHH. Both p.G18D and p.L58L (c.172C>T) were identified in heterozygous state, and the p.W275* variant was identified in two of these males, since one in homozygous and in another in heterozygous state in association with the silent variant p.L58L (c.172C>T). In silico analyses suggested that p.G18D might be damaging to the NK3R. In vitro studies of this variant (p.G18D) showed that the amount of inositol phosphate (IP) was not significantly different in cells transfected with the p.G18D mutant receptor than in cells transfected with the wild type receptor, indicating that this variant did not alter the function of the neurokinin B receptor. All new variants identified in the TAC3 and TACR3 genes were absent in 300 control alleles. In conclusion, we identified new variants in the TAC3 and TACR3 genes in Brazilian patients with idiopathic central pubertal disorders. We confirm the key role of the NKB/NK3R complex in the etiology of normosmic IHH

Avanços recentes na etiologia da puberdade precoce foram obtidos a partir da análise do genoma por sequenciamento global. Mutações inativadoras do gene MKRN3 representam uma causa importante de puberdade precoce central (PPC) familial (33-46% dos casos). O objetivo do estudo foi a análise do DNA genômico de pacientes com PPC de origem familial ou esporádica sem mutações deletérias no gene MKRN3. Foram selecionados 68 indivíduos com PPC (37 com a forma familial e 31, aparentemente, esporádicos). O DNA genômico foi extraído do sangue periférico ou da saliva dos pacientes com PPC. A técnica de sequenciamento genômico em larga escala (ILLUMNA -Clonal Single Molecule Array Technology - CSMA) foi usada na busca de novos genes implicados com o desenvolvimento puberal prematuro em seis indivíduos, sendo três afetados e três não afetados, pertencentes a uma grande família brasileira com PPC (Família 1). Mutações em um gene candidato foram pesquisadas em 64 pacientes por sequenciamento automático direto (método de Sanger). Em um subgrupo de pacientes, foi realizada a técnica de MLPA com sondas customizadas na busca de deleções. Por sequenciamento genômico global, foi identificado um novo complexo rearranjo no gene DLK1, caracterizado por uma deleção de, aproximadamente, 14.000 pb na região 5\' não traduzida (5\'UTR), englobando o início do exon 1, associada a uma duplicação de uma região do intron 3 de 269 pb. O gene DLK1 está localizado no braço longo do cromossomo 14 (14q32.2) e sofre imprinting materno. Este lócus está associado à síndrome de Temple, uma doença complexa com múltiplas manifestações, incluindo puberdade precoce central em até 90% dos casos. Para investigar o efeito dessa deleção genômica, as concentrações séricas da proteína DLK1 pelo método ELISA foram medidas nas pacientes afetadas da Família 1. Valores indetectáveis de DLK1 foram encontrados nestas pacientes. O fenótipo das pacientes afetadas da Família 1 caracterizou-se por uma PPC típica, sem sinais sindrômicos (excluída a síndrome de Temple). Posteriormente, por meio do sequenciamento direto, duas novas mutações inativadoras no gene DLK1 foram identificadas (p.Val272Cysfs*14 e p.Pro160Leufs*50) em duas famílias (Famílias 2 e 3) com PPC ou história de menarca precoce. O estudo de segregação nas Famílias 1 e 2 confirmou o padrão de herança autossômico dominante com penetrância completa e transmissão exclusiva pelo alelo paterno. A média de idade de início da puberdade nas pacientes afetadas do sexo feminino foi de 5,4 anos. A técnica de MLPA com sondas customizadas para o gene DLK1 não encontrou outras deleções no subgrupo estudado. Em conclusão, foram identificadas três mutações inativadoras no gene DLK1 associadas à PPC familial de origem paterna. O DLK1 é o segundo gene imprintado associado a distúrbios puberais em humanos. Este achado sugere um papel dos genes imprintados no controle da puberdade. O mecanismo pelo qual esse gene afeta a puberdade ainda é desconhecido / Recent advances in the etiology of precocious puberty were obtained from the whole-genome sequencing analysis. Inactivating mutations of the MKRN3 gene represent a major cause of familial central precocious puberty (CPP) (33%- 46% of the cases). The objective of the study was to analyze the genomic DNA of patients with familial or sporadic CPP without deleterious mutations in the MKRN3 gene. Sixty-eight individuals with CPP (37 with familial form and 31 apparently sporadic cases) were selected. The genomic DNA was extracted from the peripheral blood or saliva of patients with CPP. We used the whole-genomic sequencing technique (ILLUMNA - Clonal Single Molecule Array Technology - CSMA) searching for a new candidate genes implicated in premature pubertal development in 6 individuals, 3 affected and 3 non-affected, belonging to a large Brazilian family with CPP (Family 1). Mutations in one candidate gene were investigated in 64 patients through automatic sequencing (Sanger\'s method). In a subgroup of patients, MLPA using synthetic MLPA probes was performed to search for deletions. A new complex rearrangement in the DLK1 gene characterized by a deletion of approximately 14.000pb in the 5\' untranslated (5\'UTR), encompassing the start of exon 1, associated with a duplication of a region of intron 3 of 269 bp was identified by whole-genomic sequencing. The DLK1 gene is located on the long arm of chromosome 14 (14q32.2) and it is maternally imprinted gene. This locus is associated with Temple syndrome, a complex disorder with multiple alterations, including central precocious puberty in up to 90% of cases. To investigate the effect of this genomic deletion, a serum measurement of DKL1 protein using ELISA method was performed in the affected patients from Family 1. Undetectable serum DLK1 levels were found in these patients. The phenotype of affected patients from Family 1 was characterized by a typical CPP, without syndromic signs (excluding Temple syndrome). Posteriorly, two new inactivating mutations in the gene DLK1 were identified (p.Val272Cysfs*14 and p.Pro160Leufs*50) through direct sequencing in two families (Families 2 and 3) with CPP or precocious menarche history. The segregation studies in Families 1 and 2 confirmed the pattern of dominant autosomal inheritance with complete penetrance and exclusive transmission by the paternal allele. The average age of puberty onset in the affected female patients was 5.4 years. The MLPA technique with synthetic MLPA probes for the DLK1 gene did not find other deletions in the studied subgroup. In conclusion, we identified 3 paternally inherited inactivating mutations in the DLK1 gene associated with familial CPP. The DLK1 is the second imprinted gene associated with pubertal disorders in humans. This finding suggests a role of the imprinted genes in puberty control. The mechanism through which this gene affects puberty is still unknown