Kiso H, Takahashi K, Saito K, Togo Y, Tsukamoto H, et al. (2014) Interactions between BMP-7 and USAG-1 (Uterine Sensitization-Associated Gene-1) Regulate Supernumerary Organ Formations. PLoS ONE 9(5): e96938. / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18545号 / 医博第3938号 / 新制||医||1006(附属図書館) / 31445 / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 戸口田 淳也, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In vivo organogenesis is based on the temporal-spatial developmental processes that depend on cell behaviour, for example on their growth, migration, differentiation and intercellular interactions. Such behaviour is regulated by appropriate transient expression of various signalling molecules. Despite the significant advances in therapeutic strategies, the secret of the development of the biological replacement of a damaged or missing tooth has not yet been revealed. In this context, animal models provide a powerful tool for studying tooth normogenesis and pathogenesis in both basic and applied research. Early development of the tooth shares similar morphological and molecular features with other ectodermal organs. At the same time, these features are largely preserved also between species, which is advantageous for the use of model organisms. The dental formula of both: the human and the mouse are reduced against a common ancestor, but both groups of organisms evince simple as well as multicusped teeth. In both, structures called rudimentary were found. These structures are suppressed during ontogenetic development and generally they are not attributed to essential functions. That is why we aimed to study dental rudiments in detail and reveal their function in odontogenesis. This work presents new...

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Anomalias dentárias ocorrem com maior frequência em indivíduos com fissura labiopalatina (Slayton et al 2003) e em seus irmãos no afetados quando comparados à população sem fissura (Eerens et al 2001). Estudos realizados em indivíduos sem fissura demonstram que diferentes tipos de anomalias apresentam-se frequentemente associadas entre si (Baccetti 1998, Garib, Peck e Gomez 2009, Garib et al 2010). O objetivo do presente estudo foi determinar associações entre anomalias dentárias em indivíduos com fissura de lábio e palato, considerando que esse conhecimento pode fornecer informações essenciais para a definição de protocolos de tratamento. Metodologia: foram analisadas 500 radiografias panorâmicas de pacientes com fissura completa de lábio e palato unilateral, na faixa etária de 7 a 11 anos, obtidas do arquivo de Radiologia do Hospital de Reabilitação de Anomalias Craniofaciais, da Universidade de São Paulo (HRAC-USP). As radiografias foram avaliadas por um único examinador sobre um negatoscópio para o diagnóstico das anomalias dentárias. Os 30 primeiros registros foram avaliados duas vezes, com intervalo de uma semana para análise da concordância intra-examinador, com valores de Kappa acima de 0,77. As anomalias dentárias dos lados fissurado (LF) e não fissurado (LNF) foram analisadas separadamente. Os dados obtidos foram avaliados por estatística descritiva e comparados pelo Teste de McNemar. Resultados: As anomalias mais frequentemente observadas foram a agenesia (58,4%) e a microdontia (33,6%). O LF apresentou maior prevalência de anomalias dentárias (73%) quando comparado com o LNF (27%). Associações estatisticamente significativas foram observadas entre hipodontia -microdontia (p<0,001), hipodontia - dente supranumerário (p<0,001), hipodontia - transposição (p<0,001) e infraoclusão - erupço ectópica do 1o molar permanente superior (p=0,004). Conclusão: este estudo revelou associações estatisticamente significativas na ocorrência de anomalias dentárias em indivíduos com fissura labiopalatina, sugerindo que sua etiologia pode compartilhar aspectos genéticos e ambientais comuns ou estar diretamente relacionada à falta de massa mesenquimal. / Dental anomalies frequently occur in individuals with cleft lip and palate (Slayton et al 2003) and their unaffected siblings compared to the population without cleft (Eerens et al 2001). Studies in individuals without cleft demonstrate that different types of anomalies are associated with each other (Baccetti 1998, Garib Peck and Gomez, 2009, Garib et al 2010). The aim of this study was to determine associations between dental anomalies in individuals with complete cleft lip and palate, considering that this knowledge can provide essential information for defining treatment protocols. Methods: The study was conducted on 500 panoramic radiographs of patients with unilateral complete cleft lip and palate, aged 7-11 years, obtained from the Radiology files, Hospital for Rehabilitation of Craniofacial Anomalies, University of So Paulo (HRAC - USP). Radiographs were evaluated by a single examiner on a light box for the diagnosis of dental anomalies. The first 30 records were assessed twice, with a one-week interval to analyze the intra-examiner agreement, with Kappa values above 0.77. The dental anomalies were analyzed separately for the cleft side (CS) and non-cleft side (NCS). Data were analyzed by descriptive statistics and compared using McNemar test. Results: The most frequently observed abnormalities were hypodontia (58.4%) and microdontia (33.6%). The CS showed higher prevalence of dental anomalies (73%) compared to the NCS (27%). Statistically significant associations were observed between hypodontia - microdontia (p < 0.001), hypodontia - supernumerary tooth (p < 0.001), hypodontia - transposition (p < 0.001) and infraocclusion - ectopic eruption of the maxillary first permanent molar (p = 0.004). Conclusion: This study revealed significant associations in the occurrence of dental anomalies in individuals with cleft lip and palate, suggesting that its etiology may share common genetic and environmental factors or be directly related to the lack of mesenchymal mass.

Les chromosomes marqueurs surnuméraires (CMS) sont définis comme des chromosomes de structure anormale qui ne peuvent pas être identifiés ni caractérisés de façon non ambigüe par les techniques de cytogénétique conventionnelle seules et qui sont de taille égale ou plus petits qu’un chromosome 20 de la même métaphase. La prévalence de cette anomalie chromosomique est estimée à 0,071% en post-natal, 0,075% en diagnostic prénatal et 0,288% chez les patients atteints de retard mental et/ou du développement. Chez les patients infertiles, la fréquence des CMS est estimée à 0,122% et varie selon le sexe. Les CMS sont sans conséquence clinique dans 70% des cas. Dans un tiers des cas, ils peuvent être responsables de nombreuses anomalies du développement et de la reproduction humaine. A ce jour, il existe très peu d’études de caractérisation des CMS permettant une cartographie précise des gènes présents. Dans ce travail, nous avons étudié une série de huit CMS par cytogénétique conventionnelle, FISH (fluorescent in situ hybridization) et CGH array (array comparative genomic hybridization). Nous avons établi une cartographie des gènes présents dans ces CMS. L’étude de la relation génotype-phénotype des patients nous a permis de proposer l’implication de certains gènes candidats dans des anomalies du développement et de la reproduction humaine. Notre étude de l’implication des CMS dans les anomalies du développement humain s’est basée sur l’étude cytogénétique de trois fœtus. Les deux premiers fœtus étaient porteurs d’un CMS(20) en anneau. Le sujet 1 présentait un retard de croissance intra-utérin (RCIU) et une dysmorphie cranio-faciale. Le sujet 2 n’avait pas d’anomalies particulières à part une obésité diagnostiquée à l’âge de quatre mois. La taille de ces CMS(20) était de 13,6 Mb pour le sujet 1 et 4,8 Mb pour le sujet 2. Le gène SSTR4 présent sur le CMS(20) du sujet 2 code pour un récepteur de la somatostatine. Cette hormone joue un rôle dans le comportement alimentaire. Le troisième fœtus présentait un hygroma kystique et un RCIU associé à un CMS(13) néocentromérique. Les explorations par CGH array ont révélé un gain chromosomique de la région 13q21.1qter de 39 Mb contenant 80 gènes dont GPC5, GPC6, SPRY2, EFNB2, SOX1 et DZIP1. La modification d’expression de ces gènes est susceptible d’être responsable du phénotype des sujets étudiés.Notre étude de l’implication des CMS dans les anomalies de la reproduction humaine s’est basée sur l’étude cytogénétique de cinq patients qui présentaient des troubles de la fertilité (anomalies de la spermatogenèse, insuffisance ovarienne prématurée, syndrome des ovaires polykystiques et fausses couches spontanées). Les CMS explorés par CGH array correspondaient aux régions chromosomiques 15q11.2 (3,6 Mb), 21p11.2 (0,266 Mb), 6p11.2q12 (9 Mb) et 20p11.21 (3,3 Mb). Le CMS d’une des patientes ne contenait pas d’euchromatine et une autre patiente était porteuse de deux CMS d’origines chromosomiques différentes. Plusieurs gènes candidats (POTE B, BAGE et THBD) ont pu être identifiés. La modification de leur expression ainsi que des effets mécaniques ou biochimiques perturbant la méiose et la maturation des gamètes pourraient être responsables des troubles de la fertilité observés chez ces patients. L’étude des CMS par CGH array nous a permis de caractériser précisément les points de cassure des CMS, leur taille et leur composition génétique afin de cartographier les gènes présents dans les CMS et d’établir des relations entre le génotype et le phénotype des patients. / Small supernumerary marker chromosomes (sSMC) are defined as structurally abnormal chromosomes which cannot be unambiguously identified or characterized by conventional banding cytogenetic techniques alone and are generally equal in size or smaller than a chromosome 20 of the same metaphase spread. sSMC frequency is estimated at 0.071% in postnatal cases, 0.075% in prenatal cases, and 0.288% for mentally and/or development retarded patients. In infertile patients cases, sSMC frequency is estimated at 0.122% and is different in male (0.165%) and female infertility (0.022%). sSMC have no clinical consequences in 70% of the cases. In one third of the cases, they can be responsible for various human development and reproduction anomalies. To date, only a few studies precisely characterizing the sSMC contents have been performed.In this study, we used conventional cytogenetics, FISH (fluorescent in situ hybridization) and array CGH (array comparative genomic hybridization) to characterize eight sSMC and to precisely localize the genes included. The study of the genotype-phenotype correlations of the patients led us to suppose the implication of some candidate genes in human development and reproduction anomalies.Our study of the implication of sSMC in human development anomalies was based on the cytogenetic study of three fetuses. The first two fetuses carried a ring sSMC(20). Case 1 presented with intrauterine growth retardation and craniofacial dysmorphism. Case 2 had a normal phenotype except for obesity diagnosed at the age of four months. The size of these sSMC(20) was approximately 13,6 Mb for case 1 and 4,8 Mb for case 2. The SSTR4 gene located on the case 2 sSMC(20) is coding for one of the somatostatin receptor. This hormone has multiple effects on variable cells and is implicated in the regulation of food behavior, which could explain the obesity of case 2. Case 3 presented with intrauterine growth retardation and a cystic hygroma associated with a neocentric sSMC(13). Array CGH investigations showed a 32.9 Mb gain from 13q31.1 to 13qter region containing 80 genes. Among these genes, six genes could be involved in the phenotype of the proband (GPC5, GPC6, SPRY2, EFNB2, SOX1 and DZIP1). The expression modification of these genes could be responsible for the phenotype observed.Our study of the implication of sSMC in human reproduction anomalies was based on the cytogenetic study of five patients presenting fertility troubles (spermatogenesis impairment, ovarian insufficiency, polycystic ovary syndrome and repeated abortions). The sSMC explored by array CGH corresponded to the 15q11.2 region (3.6 Mb), the 21p11.2 region (0.266 Mb), the 6p11.2q12 region (9 Mb) and 20p11.21 region (3.3 Mb). The sSMC of one of the patients did not contain euchromatin and one patient carried two sSMC derived from two different chromosomes. Among the genes present on the sSMC, some candidate genes (POTE B, BAGE and THBD) have been identified. The modification of their expression and mechanical or biochemical effects of the sSMC impeding meiosis could be directly responsible for the fertility trouble observed in these patients. A detailed molecular cytogenetic investigation using array CGH allowed us to precisely characterize the chromosomal breakpoints, the size and genomic constitution of sSMC. This study may be helpful to address genotype–phenotype correlations and for medical and genetic counseling.

Οι υπερίτες του αζώτου (nitrogen mustards) συνιστούν μία αποτελεσματική ομάδα φαρμάκων που χρησιμοποιούνται στη χημειοθεραπεία του καρκίνου. Πρόσφατα ευρήματα της ερευνητικής μας ομάδας έδειξαν ότι οι υπερίτες του αζώτου μελφαλάνη (MEL), χλωραμπουκίλη (CAB) και ο δραστικός της μεταβολίτης, το PHE, επιδεικνύουν ισχυρή θραυσματογόνο δράση, αλλά επιπρόσθετα, εμφανίζουν ανευπλοειδογόνο δράση, διαταράσσοντας το χρωμοσωματικό αποχωρισμό μέσω τροποποιήσεων της δομής και λειτουργίας της μιτωτικής συσκευής. Στην παρούσα διατριβή, διερευνήθηκε περαιτέρω ο μηχανισμός της ανευπλοειδογόνου δράσης των παραπάνω δραστικών ενώσεων και πραγματοποιήθηκε σύγκριση της γενετικής δράσης δύο νέων στεροειδών αναλόγων του PHE, τα ανάλογα ΕΑ-92 και ΕΑ-97 με αυτήν των MEL, CAB και PHE. Τα στεροειδή ανάλογα σχεδιάστηκαν με στόχο την αύξηση της εκλεκτικότητας της αντινεοπλασματικής δράσης. Η ικανότητα των MEL, CAB και PHE να προκαλούν φαινόμενα χρωμοσωματικής καθυστέρησης μελετήθηκε σε σύγκριση με τα στεροειδή ανάλογα ΕΑ-92 και ΕΑ-97. Η μελέτη πραγματοποιήθηκε σε ανθρώπινα λεμφοκύτταρα in vitro με τη μέθοδο αναστολής της κυτταροκίνησης (CBMN) σε συνδυασμό με τη μέθοδο FISH και τη χρήση πανκεντρομερικού ανιχνευτή. Επιβεβαιώθηκε η θραυσματογόνος και ανευπλοειδογόνος δράση των ενώσεων MEL, CAB και PHE, ενώ φάνηκε ότι τα στεροειδή ανάλογα ΕΑ-92 και ΕΑ-97 προκαλούν αποκλειστικά χρωμοσωματική θραύση. Το φαινόμενο της χρωμοσωματικής καθυστέρησης μελετήθηκε επίσης με τη μέθοδο CREST στην κυτταρική σειρά ποντικού C2C12. Με τη μέθοδο αυτή, επιβεβαιώθηκε η διπλή γενετική δράση των ενώσεων MEL, CAB και PHE. Τα στεροειδή ανάλογα ΕΑ-92 και ΕΑ-97 εμφανίστηκαν ως οι ηπιότεροι επαγωγείς ΜΝ και προκαλούν κυρίως χρωμοσωματική θραύση, ενώ ήπια ανευπλοειδογόνο δράση παρουσίασε μόνο το ανάλογο ΕΑ-92. Ακολούθως, εξετάσθηκε η ικανότητα των υπό εξέταση χημικών ενώσεων ΕΑ-92 και ΕΑ-97, να επηρεάζουν τη δομή και λειτουργία της μιτωτικής συσκευής σε σχέση με αυτήν των ενώσεων MEL, CAB, PHE, με διπλό ανοσοφθορισμό για τη β- και γ-τουμπουλίνη. Παρατηρήθηκε ότι όλες οι ενώσεις, εκτός από το στεροειδές ΕΑ-97 προκαλούν τη δημιουργία πολυπολικών μεταφάσεων, ενώ όλες οι ενώσεις επάγουν το σχηματισμό μεσοφασικών κυττάρων με ανώμαλο αριθμό κεντροσωμάτων. Όλες οι υπό εξέταση χημικές ενώσεις εμφανίζουν κυτταροτοξικότητα και καθυστέρηση του κυτταρικού κύκλου σε καλλιέργειες ανθρώπινων λεμφοκυττάρων και στα κύτταρα ποντικού C2C12. Στη συνέχεια διερευνήθηκε η ικανότητα των υπό εξέταση ενώσεων να επάγουν την απόπτωση και μελετήθηκε ο ρόλος της απόπτωσης στην εκδήλωση της γενετικής δράσης των ενώσεων MEL, CAB και PHE. Η μελέτη αυτή πραγματοποιήθηκε στα κύτταρα C2C12 με τη μέθοδο της διπλής χρώσης Αννεξίνης V/Ιωδιούχου προπιδίου και το διπλό ανοσοφθορισμό β- και γ-τουμπουλίνης, ανεξάρτητα, σε κύτταρα ποντικού C2C12, παρουσία του γενικού αναστολέα της δράσης των κασπασών, Z-VAD-FMK, αλλά και αναστολέων της δράσης συγκεκριμένων κασπασών. Όλες οι υπό εξέταση ενώσεις επάγουν χαμηλά ποσοστά απόπτωσης. Οι κασπάσες-3, -6 και -8 συμμετέχουν στην επαγόμενη από τη MEL απόπτωση αλλά δεν συμμετέχουν στην απομάκρυνση των κυττάρων με μικροπυρήνες που επάγονται από τη δράση της ίδιας ένωσης. Η απόπτωση αποτελεί μηχανισμό απομάκρυνσης των κυττάρων με μικροπυρήνες και κανονικό κεντροσωματικό αριθμό που επάγονται από τις ενώσεις MEL, CAB και PHE. Αντίθετα, τα κύτταρα με υπεράριθμα κεντροσώματα, που προκύπτουν από τη δράση των παραπάνω ενώσεων δεν απομακρύνονται μέσω απόπτωσης. Για την περαιτέρω διερεύνηση του μηχανισμού με τον οποίο οι δραστικές ενώσεις MEL και CAB εκφράζουν τις ανευπλοειδογόνες ιδιότητες τους, μελετήθηκε η επίδραση τους στην έκφραση των πρωτεϊνών Aurora-B, survivin, Aurora-A και γ-τουμπουλίνη σε κύτταρα ποντικού C2C12, με τη μέθοδο της ανοσοαποτύπωσης των πρωτεϊνών. Παράλληλα μελετήθηκε η ένωση ΕΑ-97, η οποία σύμφωνα με τα ευρήματα μας, εμφάνισε αποκλειστικά θραυσματογόνο δράση. Οι ενώσεις MEL και CAB, εκδηλώνουν τις ανευπλοειδογόνες ιδιότητες τους προκαλώντας μείωση της έκφρασης των πρωτεϊνών Aurora-B και survivin και επάγοντας την αύξηση της έκφρασης της πρωτεΐνης Aurora-A. Επιπρόσθετα, η ένωση MEL, προκαλεί αύξηση της έκφρασης της γ-τουμπουλίνης. Τα ευρήματα αυτά υποδεικνύουν τη συμμετοχή των παραπάνω πρωτεϊνών στην εκδήλωση της ανευπλοειδογόνου δράσης των ενώσεων που μελετήθηκαν. Αντίθετα το στεροειδές ανάλογο ΕΑ-97 που εμφανίζει αποκλειστικά θραυσματογόνο δράση, δεν μεταβάλλει την έκφραση των παραπάνω πρωτεϊνών. / Nitrogen mustards represent an effective class of drugs that are used in chemotherapy. Recent findings of our group have shown that nitrogen mustard analogues, melphalan (MEL), chlorambucil (CAB) and PHE, in addition to their clastogenic activity, they exert their aneugenic potential by affecting chromosome segregation due to modifications of mitotic apparatus. In the present study, we investigated the mechanism by which the above compounds display their aneugenicity in comparison with two new steroidal analogues of PHE, EA-92 and EA-97, which were designed aiming at most effective antineoplasmatic activity. The ability of MEL, CAB and PHE to induce chromosome delay events was studied in comparison with the steroidal analogues EA-92 and EA-97. The mechanism of micronucleation was determined by Cytokinesis Block Micronucleus assay (CBMN assay) in combination with Fluorescence In Situ Hybridization (FISH) using pancentromeric DNA probe. It was confirmed that MEL, CAB and PHE generated MNi by two mechanisms, chromosome breakage and chromosome delay, while EA-92 and EA-97 induced the formation of MN originated exclusively from chromosome breakage events. The ability of the tested compounds to induce chromosome delay was also investigated in C2C12 mouse cells by CREST analysis. The dual genetic activity of MEL, CAB, and PHE was confirmed in a different biological system. The analogues EA-92 and EA-97 appeared as weaker MN inducers and they induced mainly chromosome breakage, while a weak aneugenic activity was observed for EA-92. The ability of the nitrogen mustard analogues to affect the organization of mitotic apparatus was investigated in comparison with MEL, CAB and PHE by double immunofluorescence of β- and γ-tubulin in C2C12 mouse cells. It was observed that all compounds, except EA-97, induced mutlipolar metaphases, and also generated interphase cells with abnormal centrosome number. All compounds displayed increased cytotoxicity and they caused cell cycle delay in human lymphocyte cultures and in C2C12 mouse cells. The ability of the tested compounds to induce apoptosis was studied by Annexin V/PI assay. It was revealed that all compounds induced apoptosis. The effect of apoptosis on the genetic activity of MEL, CAB and PHE was investigated by inhibition of apoptosis in the presence of the inhibitor Z-VAD-FMK and the use of specific inhibitors for caspase -3, -6, -8 and -1. For this reason Annexin V/PI assay and double immunofluorescence of β- and γ-tubulin were performed, independently in C2C12 mouse cells. Caspases -3, -6 and -8 are involved in melphalan-induced apoptosis, but they are not involved in the elimination of cells in the presence of melphalan. Apoptosis is the responsible mechanism for the exclusion of cells with MNi and normal centrosome number that are induced by MEL, CAB and PHE. On the contrary, cells exerting supernumerary centrosomes are not eliminated by apoptosis in the presence of the above compounds. To further elucidate the mechanisms by which MEL and CAB exert their aneugenic potential, we examined the ability of the compounds to alter the expression of proteins having important role in chromosome segregation, such as the proteins Aurora-B, survivin, Aurora-A and γ-tubulin. The analysis was performed by Western blot method in C2C12 mouse cells. We also studied the steroid analogue EA-97, which according to our findings acts as a pure clastogen and do not exert aneugenic potential as opposed to MEL and CAB. MEL and CAB exert their aneugenic potential by the reduction of Aurora-B and survivin expression and by enhancing the expression of Aurora-A. γ-tubulin was upregulated in the presence of MEL. These findings show the implication of these proteins in chromosome delay events induced by MEL and CAB. On the other hand, the analogue EA-97 did not affect the expression of the above proteins.

Submitted by Luciana Sebin (lusebin@ufscar.br) on 2016-10-11T19:38:10Z No. of bitstreams: 1 DissDPMcg.pdf: 1163116 bytes, checksum: 38b9e1756b3619ed64928eaf8458e248 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-10-17T13:04:52Z (GMT) No. of bitstreams: 1 DissDPMcg.pdf: 1163116 bytes, checksum: 38b9e1756b3619ed64928eaf8458e248 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2016-10-17T13:05:00Z (GMT) No. of bitstreams: 1 DissDPMcg.pdf: 1163116 bytes, checksum: 38b9e1756b3619ed64928eaf8458e248 (MD5) / Made available in DSpace on 2016-10-17T13:13:15Z (GMT). No. of bitstreams: 1 DissDPMcg.pdf: 1163116 bytes, checksum: 38b9e1756b3619ed64928eaf8458e248 (MD5) Previous issue date: 2016-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Partamona (Schwarz, 1939) is a stingless bee genus which has as an interesting trait, the presence of B chromosomes in some species. These chromosomes were identified in six closely related species so far, all included in the cupira group, except P. helleri, which has an uncertain position in Partamona phylogeny, but it is possibly related to this group. We analyzed 894 colonies from 128 locations of thirteen species, using a SCAR marker specific for Partamona B chromosomes. The results enabled an increase in knowledge about species number with supernumerary chromosomes in this genus, including species from the pearsoni clade. The high number of colonies analyzed allowed to set the B chromosomes frequency on carrier species. A significant positive correlation between latitude and B chromosomes frequency was found in P. helleri, with higher frequency in north decreasing towards the south of the species distribution. The B chromosome transmission rate was estimated for three species that showed the highest frequencies of these chromosomes (P. helleri, P. cupira and P. rustica). The result was as expected for a regular meiotic behavior, which queens with one supernumerary chromosome have 50% chance to transfer the B chromosome to the offspring, suggesting an absence drive in these species. The Sequences obtained using SCAR primers had high quality and were different from those previously reported in another studies, showing no undefined sites, indicating greater effectiveness for sequencing using the internal primers designed in our laboratory. The comparison between obtained sequences revealed a high similarity between species, with only two haplotypes and no intraspecific differences, suggesting a single origin for Partamona B chromosomes. The sequence obtained for P. helleri was different from that obtained for the cupira clade species, suggesting that P. helleri did not transmit the B chromosomes to the other species, as previously suggested. The high frequency in some species and the presence in the pearsoni clade suggest a more complex 4 evolutionary history and an older origin than previously thought for Partamona B chromosomes. / Partamona (Schwarz, 1939) é um gênero de abelhas sem ferrão que apresenta como interessante característica a presença de cromossomos B em algumas espécies. Até o momento, esses cromossomos haviam sido identificados em seis espécies intimamente relacionadas, todas incluídas no clado cupira, e P. helleri, de posição incerta na filogenia de Partamona, mas possivelmente relacionada a este clado. A análise de 894 colônias provenientes de 128 localidades de 13 espécies do gênero, utilizando um marcador SCAR (Sequence Characterized Amplified Region) específico para os cromossomos B de Partamona, permitiu ampliar o conhecimento do número de espécies do gênero portadoras destes cromossomos, incluindo espécies do clado pearsoni. A partir dos resultados desta análise foi possível também, definir a frequência dos cromossomos B nas espécies portadoras. Em P. helleri foi encontrada uma correlação positiva significativa entre latitude e frequência de cromossomos B, com maior frequência no norte e diminuição em direção ao sul da distribuição da espécie. A taxa de transmissão de cromossomos B foi estimada para as três espécies em que estes cromossomos foram mais frequentes (P. helleri, P. cupira e P. rustica). O resultado foi consistente com o esperado para um comportamento meiótico regular na transmissão dos cromossomos B da rainha para a prole, sugerindo que nessas espécies não ocorra um desvio meiótico que favoreça a transmissão. As sequências obtidas com os primers SCAR apresentaram alta qualidade e foram diferentes das sequências reportadas na literatura, não apresentando sítios indefinidos, apontando para uma maior acurácia de sequenciamento com o uso dos primers internos desenhados em nosso laboratório. A comparação entre as sequências revelou uma alta similaridade entre espécies, com apenas dois haplótipos e ausência de diferenças intraespecíficas, sugerindo uma origem única para os cromossomos B de Partamona. A sequência obtida para P. helleri foi diferente da sequência obtida para as 2 outras espécies do clado cupira, sugerindo que essa espécie não transmitiu os cromossomos B para as outras espécies do grupo, como previamente sugerido. A alta frequência de cromossomos B em algumas espécies do gênero e a presença de cromossomos B em espécies do clado pearsoni sugere uma história evolutiva mais complexa e uma origem mais antiga do que se presumia anteriormente para os cromossomos B de Partamona.

Anomalias dentárias ocorrem com maior frequência em indivíduos com fissura labiopalatina (Slayton et al 2003) e em seus irmãos no afetados quando comparados à população sem fissura (Eerens et al 2001). Estudos realizados em indivíduos sem fissura demonstram que diferentes tipos de anomalias apresentam-se frequentemente associadas entre si (Baccetti 1998, Garib, Peck e Gomez 2009, Garib et al 2010). O objetivo do presente estudo foi determinar associações entre anomalias dentárias em indivíduos com fissura de lábio e palato, considerando que esse conhecimento pode fornecer informações essenciais para a definição de protocolos de tratamento. Metodologia: foram analisadas 500 radiografias panorâmicas de pacientes com fissura completa de lábio e palato unilateral, na faixa etária de 7 a 11 anos, obtidas do arquivo de Radiologia do Hospital de Reabilitação de Anomalias Craniofaciais, da Universidade de São Paulo (HRAC-USP). As radiografias foram avaliadas por um único examinador sobre um negatoscópio para o diagnóstico das anomalias dentárias. Os 30 primeiros registros foram avaliados duas vezes, com intervalo de uma semana para análise da concordância intra-examinador, com valores de Kappa acima de 0,77. As anomalias dentárias dos lados fissurado (LF) e não fissurado (LNF) foram analisadas separadamente. Os dados obtidos foram avaliados por estatística descritiva e comparados pelo Teste de McNemar. Resultados: As anomalias mais frequentemente observadas foram a agenesia (58,4%) e a microdontia (33,6%). O LF apresentou maior prevalência de anomalias dentárias (73%) quando comparado com o LNF (27%). Associações estatisticamente significativas foram observadas entre hipodontia -microdontia (p<0,001), hipodontia - dente supranumerário (p<0,001), hipodontia - transposição (p<0,001) e infraoclusão - erupço ectópica do 1o molar permanente superior (p=0,004). Conclusão: este estudo revelou associações estatisticamente significativas na ocorrência de anomalias dentárias em indivíduos com fissura labiopalatina, sugerindo que sua etiologia pode compartilhar aspectos genéticos e ambientais comuns ou estar diretamente relacionada à falta de massa mesenquimal. / Dental anomalies frequently occur in individuals with cleft lip and palate (Slayton et al 2003) and their unaffected siblings compared to the population without cleft (Eerens et al 2001). Studies in individuals without cleft demonstrate that different types of anomalies are associated with each other (Baccetti 1998, Garib Peck and Gomez, 2009, Garib et al 2010). The aim of this study was to determine associations between dental anomalies in individuals with complete cleft lip and palate, considering that this knowledge can provide essential information for defining treatment protocols. Methods: The study was conducted on 500 panoramic radiographs of patients with unilateral complete cleft lip and palate, aged 7-11 years, obtained from the Radiology files, Hospital for Rehabilitation of Craniofacial Anomalies, University of So Paulo (HRAC - USP). Radiographs were evaluated by a single examiner on a light box for the diagnosis of dental anomalies. The first 30 records were assessed twice, with a one-week interval to analyze the intra-examiner agreement, with Kappa values above 0.77. The dental anomalies were analyzed separately for the cleft side (CS) and non-cleft side (NCS). Data were analyzed by descriptive statistics and compared using McNemar test. Results: The most frequently observed abnormalities were hypodontia (58.4%) and microdontia (33.6%). The CS showed higher prevalence of dental anomalies (73%) compared to the NCS (27%). Statistically significant associations were observed between hypodontia - microdontia (p < 0.001), hypodontia - supernumerary tooth (p < 0.001), hypodontia - transposition (p < 0.001) and infraocclusion - ectopic eruption of the maxillary first permanent molar (p = 0.004). Conclusion: This study revealed significant associations in the occurrence of dental anomalies in individuals with cleft lip and palate, suggesting that its etiology may share common genetic and environmental factors or be directly related to the lack of mesenchymal mass.

RIPPLY2 is an essential part of the formation of somite patterning during embryogenesis and in establishment of rostro-caudal polarity. Here, we describe three individuals from two families with compound-heterozygous variants in RIPPLY2 (NM_001009994.2): c.238A > T, p.(Arg80*) and c.240-4 T > G, p.(?), in two 15 and 20-year-old sisters, and a homozygous nonsense variant, c.238A > T, p.(Arg80*), in an 8 year old boy. All patients had multiple vertebral body malformations in the cervical and thoracic region, small or absent rib involvement, myelopathies, and common clinical features of SCDO6 including scoliosis, mild facial asymmetry, spinal spasticity and hemivertebrae. The nonsense variant can be classified as likely pathogenic based on the ACMG criteria while the splice variants must be classified as a variant of unknown significance. With this report on two further families, we confirm RIPPLY2 as the gene for SCDO6 and broaden the phenotype by adding myelopathy with or without spinal canal stenosis and spinal spasticity to the symptom spectrum.