The aim of this study is to provide information regarding the development of aDNA research. The essay also strives to distinguish eventual impact on previous and contemporary conceptions concerning Neanderthal and modern human genetic divergence and possible interbreeding between the two species. Relatively recent Neanderthal genetic research will provide a glimpse into future explorations. Hominin species, other than Neanderthals, will be excluded from this paper as well as morphological studies and modern human evolution theories. Nature, Cell, Science, American anthropologist are examples of important publications constituting the study’s source material generated through the mandatory literature search. Genetic data results reveal genetic divergence between Neanderthals and modern humans approximately around 270 000 to 440 000 years ago or 550 000 to 765 000 years ago. The estimations for genetic divergence continue to alter with the advent of additional data. Contemporaneous genomic data results show interbreeding between Neanderthals and modern humans which contradict some prior perceptions surrounding the subject. New engendered Neanderthal genomic data indicate further exploration concerning the Neanderthal inherited genes effect on modern day human biology and physiology. / Syftet med denna studie är att delge informativ upplysning kring utvecklingen av aDNA forskning. Uppsatsen strävar även efter att urskilja eventuell påverkan på föregående och nuvarande uppfattningar angående genetisk splittring mellan Neandertalare och moderna människor och möjlig korsning mellan de två arterna. Relativt ny genetisk Neandertal forskning kommer att ge en inblick i framtida utforskningar. Andra homininer, undantagsvis för Neandertalarna, kommer att exkluderad från denna uppsats liksom morfologiska studier och teorier kring den moderna människans evolution. Nature, Cell, Science, American anthropologist är exempel på viktiga publiceringar som utgör studiens källmaterial vilket genererats genom den obligatoriska litteratursökningen. Genetiska data resultat visar genetisk splittring mellan Neandertalare och moderna människor proximalt kring 270 000 till 440 000 år sedan eller 550 000 till 765 000 år sedan. Beräkningarna för genetisk splittring ändras kontinuerligt på grund av ankomsten av nytillkommande data. Nya genomiska data resultat visar korsning mellan Neandertalare och moderna människor vilket motsäger en del föregående föreställningar kring ämnet. Ny framställd Neandertal genomiska data indikerar vidare utforskning av de ärvda Neandertal genernas möjliga påverkan på den moderna människans biologi och fysiologi.

<p>Heterosis, or hybrid vigor, is a phenomenon observed in both plant and animal systems where hybrid offspring perform better when compared to their parents. For hybrid plants, this can result in increased biomass, crop yields, and vigor when compared to the inbred parents. Even though heterosis has been used in agriculture for over a century, the molecular mechanisms that result in hybrid vigor remain elusive even after years of investigation. A molecular understanding of heterosis is desirable because it will speed up the process of breeding compatible inbred lines for developing hybrid seeds, and it will provide us with the knowledge to potentially engineer inbred lines that can mimic the beneficial phenotypic effects of heterosis, eliminating the need for farmers to buy new hybrid seeds every year. The goal of this research project is to identify genes that are required for heterotic phenotypes in maize. Our working hypothesis is that a mutation in genes that are essential for heterosis will cause an altered heterotic phenotype in hybrid maize plants. To test this hypothesis, we applied combined approaches of EMS mutagenesis, trait phenotyping in field and controlled conditions, bulk segregant analysis, whole genome sequencing, and bioinformatics analysis. First, we applied a forward genetics approach to identify mutant hybrids with altered heterosis and detected potential causal genes <em>via</em> whole genome sequencing. We identified one mutation occurring in a protein coding gene (gene ID <em>Zm00001eb305590</em>) located in a region of interest on chromosome 7, whose genotypes across various samples assayed fit the observed segregation pattern of hybrid traits. This mutation leads to a moderate or high-level codon change, indicating that this gene may play a role in mediating heterosis in maize. By investigating this gene with further studies, the learned knowledge could speed up the process of hybrid maize breeding by selecting compatible inbred lines through sequencing or by engineering hybrids that have favorable alleles for this gene.</p>

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Development of fungicide resistance in fungal and oomycete pathogens is a serious problem in grape production. Quinoxyfen is a fungicide widely used against grape powdery mildew (Erysiphe necator). In 2013, E. necator isolates with reduced quinoxyfen sensitivity (designated as quinoxyfen lab resistance or QLR) were detected in Virginia. Field trials were conducted in 2014, 2015, and 2016 at the affected vineyard to determine to what extent quinoxyfen might still contribute to disease control. Powdery mildew control by quinoxyfen was good, similar to, or only slightly less, than that provided by myclobutanil and boscalid in all three years. The frequency of QLR in vines not treated with quinoxyfen declined only slowly over the three years, from 65% to 46%. Information about the mode of action of quinoxyfen is limited; previous research suggests that quinoxyfen interferes with the signal transduction process. We profiled the transcriptomes of QLR and sensitive isolates in response to quinoxyfen treatment, providing support for this hypothesis. Additional transcriptional targets of quinoxyfen were revealed to be involved in the positive regulation of the MAPK signaling cascade, pathogenesis, and sporulation activity. Grape downy mildew (Plasmopara viticola), another important grape pathogen, is commonly controlled by phosphite fungicides. A field trial and laboratory bioassays were conducted to determine whether P. viticola isolates from vineyards with suspected control failures showed reduced sensitivity against phosphite fungicides. Prophyt applied at 14-day intervals under high disease pressure provided poor downy mildew control in the field. Next-generation sequencing technologies were utilized to identify 391,930 single nucleotide polymorphisms (SNPs) and generated a draft P. viticola genome assembly at ~130 megabase (Mb). Finally, field isolates of P. viticola collected from a Virginia vineyard with suspected mandipropamid control failure were bioassayed. The EC50 values of the isolates were >240 μg.ml-1 for mandipropamid, well above the field rate. The PvCesA3 gene of two resistant isolates was sequenced revealing that these isolates had a GGC-to-AGC substitution at codon 1105, the same mutation that has been found associated with CAA resistance elsewhere. / PHD

Salmonella est un agent pathogène animal et zoonotique d'importance mondiale. Les infections causées par cette bactérie chez les animaux d’élevage peuvent être asymptomatiques ou se traduire par une gastro-entérite ou une maladie invasive. Chez les humains, les maladies d’origines alimentaires associées à Salmonella sont principalement causées par l'ingestion de produits d'origine animale contaminés. Cependant, aucune étude n’a déterminé la prévalence ou caractérisé le profil de virulence et d’antibiorésistance de Salmonella provenant des fermes ovines du Québec, alors que cette province détient le 2ème plus grand cheptel ovin canadien. Ainsi, cette étude visait à estimer la prévalence des fermes ovines positives à Salmonella dans la province du Québec, à identifier les sérotypes circulant dans ces troupeaux, à caractériser le profil de virulence et de résistance aux antimicrobiens de souches de Salmonella isolées dans ces fermes et à évaluer l'influence de certains facteurs de risque (ex. la saisonnalité, la taille des troupeaux et le type de production) sur la prévalence de fermes ovines positives à Salmonella au Québec. Pour cela, 61 fermes ovines du Québec ont été sélectionnées aléatoirement et ont été échantillonnées. Un pool fécal provenant de 10 animaux a été prélevé par ferme. Des milieux sélectifs ont été utilisés pour isoler Salmonella dans chaque échantillon. La confirmation de Salmonella a été faite par des tests biochimiques suivis par la détection du gène invA par PCR et les sous-espèces ont été identifiées par PCR multiplex. Le rapport de prévalence a été calculé pour évaluer l’association entre les facteurs de risque et la présence de Salmonella dans les fermes. Le séquençage du génome complet de 76 isolats a permis d’identifier les sérotypes et de déterminer le profil de virulence et de résistance génotypique de ces isolats. La confirmation de la résistance phénotypique de différentes souches a été effectuée en utilisant la méthode de diffusion en milieu solide (antibiogramme) et la méthode de dilution en milieu liquide. Les résultats indiquent que 83,6% (95% CI 74,3%-92,9%) des troupeaux sont porteurs de Salmonella. Le sérotype Salmonella enterica sous-espèce diarizonae 61 k:1, 5, (7) (Sheep associated Salmonella diarizonae (SASd) a été le seul sérotype identifié dans tous les isolats qui ont été séquencés. Aucun des facteurs de risque évalués n’était associée à la présence de Salmonella. En outre, aucun gène de résistance aux antimicrobiens n’a été identifié à partir de l’analyse des génomes séquencés et toutes les souches ont été révélées comme étant phénotypiquement sensibles aux différents antimicrobiens testés. De plus, des facteurs de virulence, comme les ilots de pathogénicité (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9 et SPI-13) et le plasmide (IncX1), ont été identifiés chez les 84 SASd. Cette étude est la première à rapporter que la prévalence des fermes ovines positives à Salmonella est élevée au Québec (83,6%). Seule la présence du sérotype 61 k:1, 5, (7) a été détectée. Finalement, toutes les souches de Salmonella isolées dans le cadre de cette étude étaient sensibles aux antimicrobiens testés. / Salmonella is a world-known animal and zoonotic pathogen. Infections caused by this bacterium in livestock can be asymptomatic or result in gastroenteritis or an invasive disease. In humans, food-related diseases associated with Salmonella are mainly caused by the ingestion of contaminated products of animal origin. However, no studies have determined the prevalence or characterized the virulence and antimicrobial resistance (AMR) profiles of Salmonella from sheep farms in Quebec, where the province holds the second largest sheep population in Canada. This study aimed to estimate the prevalence of Salmonella-positive sheep farms in the province of Quebec, to identify the serotypes circulating in these flocks, to characterize the virulence and AMR profiles of Salmonella strains isolated from these farms and to assess the influence of some potential risk factors on the prevalence of Salmonella-positive sheep farms in the province of Quebec. For this purpose, 61 sheep farms in Quebec were randomly selected and sampled. A fecal pool including samples from 10 animals was collected per farm. Selective culture media were used to isolate Salmonella in each sample. Salmonella was confirmed by biochemical tests followed by the detection of invA gene by PCR and the subspecies were identified by multiplex PCR. The prevalence ratio was calculated to assess the association between risk factors and the presence of Salmonella in farms. The Whole genome sequencing (WGS) of 76 isolates was used to determine the serotypes, the virulence and the AMR profile of these isolates. The confirmation of the phenotypic AMR of these isolates (n=76) was carried out using the Kirby–Bauer disk diffusion method (antibiogram) or the broth microdilution method. The results indicated that 83.6% (95% CI 74.3%-92.9%) of the flocks are carrier of Salmonella and only the Salmonella enterica subspecies diarizonae serotype 61: k: 1, 5, (7) (sheep associated S. diarizonae, SASd) was identified. None of the tested risk factors was associated with Salmonella positivity at the flock level. In addition, no AMR genes were identified from the WGS data, and all strains were phenotypically sensitive to the various antimicrobials tested. In addition, virulence factors such as pathogenicity islands (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9 and SPI-13) and plasmid (IncX1) have been identified in the sequenced strains. This study was the first to report a high prevalence of Salmonella-positive sheep farms in Quebec (83.6%). Only the presence of serotype 61 k:1, 5, (7) was detected. Interestingly, all Salmonella strains isolated in this study were sensitive to the tested antimicrobials.

O bocavirus humano (HBoV) é um parvovirus recentemente identificado em associação com a presença de sintomas de infecção do trato respiratório. Esse vírus possui um genoma de aproximadamente 5217 nucleotídeos que contém 3 open reading frames que codificam 4 proteínas (NS1, NP-1, VP-1 e VP-2). HBoV tem sido detectado em amostras respiratórias de diversas partes do mundo, incluindo Austrália, América do Norte, Europa, Ásia e África, o que sugere uma distribuição global desse vírus. Entretanto, nenhum estudo longitudinal de HBoV em amostras respiratórias foi realizado na América Latina. Dessa forma, nós realizamos um estudo prospectivo de HBoV em lavados nasofaríngeos (LFNs) coletados de pacientes com sintomas de infecção do trato respiratório (IRA) atendidos em um hospital universitário de Ribeirão Preto, SP e em um hospital universitário de Salvador, BA no período entre 2005 a 2007. 1288 LFNs de 1217 pacientes foram encaminhados ao laboratório de virologia e foram testados por PCR para HBoV. Desses pacientes, 962 eram menores de 5 anos e 177 eram maiores de 5 anos. Além disso, também foram analisados 50 LFNs de crianças menores de 5 anos que não tinham sintomas respiratórios. Todas as amostras positivas para HBoV foram testadas para todos os outros vírus respiratórios, incluindo o vírus sincicial respiratório (HRSV), rinovirus humano (HRV), influenza humano (HFLU), metapneumovirus humano (HMPV), parainfluenza humano (HPIV), coronavirus humano (HCoV) e adenovirus humano (HAdV). A carga viral de HBoV foi determinada por PCR em tempo real em todas as amostras positivas e o genoma completo de 19 amostras de HBoV foi seqüenciado. Com intuito, de fazer um levantamento sorológico e determinar sítios replicativos de HBoV, nós ainda clonamos e expressamos em S. cerevisae (Y258) o gene de VP2, que codifica uma das proteínas do capsídeo viral. A prevalência desse vírus foi de 4,8% em crianças menores de cinco anos e de 1% em pacientes maiores de cinco anos. HBoV não foi detectado em crianças sem sintomas. Dos 259 pacientes analisados em 2005, 25 (10%) foram positivos para HBoV. Esse vírus circulou mais frequentemente em abril, mês de maior incidência do HRSV. Em 2006, HBoV foi detectado em apenas 10 LFNs de 334 (3%) amostras testadas, sem qualquer pico de freqüência. Em 2007 HBoV foi detectado em 13 de 552 (2%) amostras, com uma freqüência de detecção um pouco maior em junho e julho. Os sintomas mais comumente observados foram rinorréia, tosse, febre e chiado, que foram observados geralmente em mais de 50% dos casos positivos para HBoV. Não houve uma diferença significativa na prevalência desses sintomas entre as crianças positivas e negativas para HBoV. Entretanto, foi observada uma maior freqüência de diarréia entre as crianças com esse vírus. Nesse estudo também foi documentado uma alta freqüência de co-infecções virais entre os pacientes com HBoV. Os vírus mais frequentemente associados com o bocavirus humano foram: HRSV, HRV e HAdV. Além disso, foi detectado uma maior carga viral media e uma maior freqüência de diarréia nos 15 pacientes com infecção exclusiva por HBoV do que nos pacientes com co-infecção. Esses resultados mostraram que HBoV pode alcançar títulos enormes (tão grandes como1014/mL) em LFNs de pacientes com sintomas respiratórios e que isso é associado a de diarréia. O seqüenciamento do genoma inteiro de HBoV realizado nesse estudo indica que a divergência genômica entre as amostras desse vírus é muito pequena. Como conclusão, nós demonstramos que HBoV circula e é detectado em associação com sintomas de infecção respiratória e diarréia no Brasil. Novos estudos, com um longo acompanhamento em diferentes populações serão necessários para determinar a sazonalidade e o real impacto clínico de HBoV em nosso país. / Human bocavirus (HBoV) is a parvovirus recently identified in association with respiratory tract infections. HBoV 5217 nt genome contains 3 open reading frames encoding four proteins (NS1, NP-1, VP-1 and VP-2). HBoV has been reported in respiratory samples from children in several parts of the world (including Australia, North America, Europe, Asia, and Africa), suggesting that the virus circulates worldwide. However, no longitudinal studies of HBoV in respiratory samples have been reported in Latin America. We report a prospective study of HBoV in nasopharyngeal aspirates (NPAs) collected from patients seen for acute respiratory tract infections (ARI) at the University of Sao Paulo Hospital in Ribeirao Preto, southeast Brazil and at the University Hospital in Salvador, Brazil. 1288 NPAs from 1217 patients was submitted to the virology lab for respiratory virus detection from 2005 to 2007 and were screened for HBoV by polymerase chain reaction (PCR), whom 962 were under 5 years of age and 177 were older than 5 years. In addition, NPAs from 50 children under 12 years without IRA was also tested to HBoV for PCR. All samples positive of HBoV was tested for others respiratory virus, including the human respiratory syncitial virus (HRSV), human rhinovirus (HRV), human influenza (HFLU), human metapneumovirus (HMPV), human parainfluenza virus (HPIV), human coronavirus (HCoV) and human adenovirus (HAdV). These samples had their HBoV viral load determined by real time PCR and the viral entire genome of nineteen HBoV sample was sequenced. We also cloned and expressed in S. cerevisae (Y258) the gene of VP2, one protein of viral capside. The prevalence of this virus was of 4,8% in children under 5 years and 1% in adults, both with IRA. HBoV was not found on the patients without symptoms. In 2005, of the 259 patients tested, 25 (10%) were positive for HBoV. Interestingly, the virus circulated more frequently in April, the month of peak activity of respiratory HRSV. In 2006 HBoV was detected in only 10 NPAs out of 334 samples (3%) tested, without any notable peak of frequency. In 2007 HBoV was detected in 13 out of 552 (2%) tested samples with little higher frequency of detection in June an July. Rhinorrhea, cough, and wheezing were observed in more than 50% of the HBoV-positive children, and no obvious respiratory clinical differences were noted between HBoV-positive and negative children. However, was noted a higher frequency of diarrhea on HBoV-positive patients. In this study was also observed a larger frequency (71%) of viral coinfections between the HBoV-positive patients. The respiratory viruses more frequently associated with human bocavirus were: HRSV, HRV and HAdV. Interestingly, on the 15 HBoV-alone patients was observed a higher viral load and a higher prevalence of diarrhea than HBoV-coinfection patients. These results showed that this virus can reach enormous titles (like 1014) in NPAs from patients with respiratory infection symptoms and this is associated with diahhrea. The entire genome sequencing of HBoV of our study indicates that the genetic divergence between the HBoV lineages is small. In conclusion, we demonstrated that HBoV circulates and is detected in association with respiratory symptoms and diarrhea in Brazil. Long term surveillance will be needed to determine whether or not an HBoV season occurs and what is the real clinical impact of this virus in our country.

O objetivo deste trabalho foi investigar a presença de bactérias produtoras de &#946;-lactamases adquiridas na microbiota Gram-negativa comensal de humanos e animais domésticos em uma comunidade remota na região da Floresta Amazônica. De março a julho de 2013 foram coletadas amostras de fezes de indivíduos atendidos e funcionários de um centro assistencial em saúde restrito a comunidades indígenas e de swab retal de animais de companhia da comunidade. Nas amostras de humanos foram detectados isolados de Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter kobei e Morganella morganii, carregando os genes blaTEM-1, blaCTX-M-15, blaCTX-M-14, blaCTX-M-8 e blaGES-5. Nas amostras de animais foram detectados apenas isolados de E. coli carregando os genes blaTEM-1, blaCTX-M-14, blaCTX-M-2 e blaCTX-M-8. Foi observada a relação clonal entre isolados de E. coli de origem humana e de origem animal. Estes resultados demonstram a disseminação de um problema endêmico em áreas urbanas para uma comunidade, em teoria, com baixa exposição a antibacterianos. / The aim of this study was to investigate the presence of acquired &#946;-lactamase in the commensal Gram-negative microbiota of humans and domestic animals of a remote community in the Amazon Forest region. From March to July 2013 stool samples were collected from individuals attended in a health care center restricted to indigenous communities and from the local staff, and rectal swab samples were collected from companion animals in the community. In the human samples were detected Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter kobei and Morganella morganii isolates harboring blaTEM-1, blaCTX-M-15, blaCTX-M-14, blaCTX-M-8 e blaGES-5 genes. In the animal samples only E. coli strains harboring blaTEM-1, blaCTX-M-14, blaCTX-M-2 and blaCTX-M-8 were detected. The clonal relatedness between E. coli strains from human and animal samples was observed. These results demonstrate the dissemination of an urban endemic problem to a community, in theory, with low antimicrobial exposure.

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