Placental hydroxymethylation vs methylation at the imprinting control region 2 on chromosome 11p15.5

In addition to methylated cytosines (5-mCs), hydroxymethylcytosines (5-hmCs) are present in CpG dinucleotide-enriched regions and some transcription regulator binding sites. Unlike methylation, hydroxymethylation does not result in silencing of gene expression, and the most commonly used methods to study methylation, such as techniques based on restriction enzymatic digestion and/or bisulfite modification, are unable to distinguish between them. Genomic imprinting is a process of gene regulation where only one member of an allelic pair is expressed depending on the parental origin. Chromosome 11p15.5 has an imprinting control region (ICR2) that includes a differentially methylated region (KvDMR1) that guarantees parent-specific gene expression. The objective of the present study was to determine the presence of 5-hmC at the KvDMR1 in human placentas. We analyzed 16 third-trimester normal human placentas (chorionic villi). We compared two different methods based on real-time PCR after enzymatic digestion. The first method distinguished methylation from hydroxymethylation, while the other method did not. Unlike other methylation studies, subtle variations of methylation in ICRs could represent a drastic deregulation of the expression of imprinted genes, leading to important phenotypic consequences, and the presence of hydroxymethylation could interfere with the results of many studies. We observed agreement between the results of both methods, indicating the absence of hydroxymethylation at the KvDMR1 in third-trimester placentas. To the best of our knowledge, this is the first study describing the investigation of hydroxymethylation in human placenta using a genomic imprinting model.

Relação entre polimorfismo do gene do hormônio do crescimento e características de precocidade em novilhas da raça Nelore / Relationship between polymorphism of growth hormone and precocity traits in Nellore heifers

Avaliaram-se as relações entre o polimorfismo do gene do hormônio do crescimento (GH) e as características de precocidade, em novilhas da raça Nelore. Amostras de sangue periférico foram obtidas de 181 animais de três rebanhos distintos do estado da Bahia, nas quais foi realizada a extração de DNA e a amplificação por PCR, seguidas por digestão com enzima de restrição AluI. Os fragmentos resultantes da digestão enzimática foram analisados em gel de agarose 2 por cento para determinação dos respectivos genótipos. A frequência do alelo Leu nas amostras analisadas foi estimada em 100 por cento. Em decorrência da alta incidência de homozigose para o alelo Leu, sugere-se que o restriction fragment lenght polymorphism AluI do gene GH não possa ser considerado como marcador molecular para precocidade sexual em novilhas Nelore nesses rebanhos.

The relationships between polymorphism of growth hormone gene (GH) and precocity traits in Nellore heifers were evaluated. A total of 181 animals from three different farms of Bahia state, Brazil, were blood sampled. The DNA of each animal was extracted, amplified by PCR, and digested by "AluI" restriction enzyme, and the resultant fragments were analyzed in 2 percent agarose gel for genotype identification. The frequency of Leu allele in the analyzed samples was estimated in 100 percent. Due to the high incidence of homozygose for the Leu allele, it is suggested that the restriction fragment lenght polymorphism AluI of GH gene can not be considered as a molecular marker for sexual precocity in Nellore heifers of those herds.

Cryptic mosaicism involving a second chromosome X in patients with Turner syndrome

The high abortion rate of 45,X embryos indicates that patients with Turner syndrome and 45,X karyotype could be mosaics, in at least one phase of embryo development or cellular lineage, due to the need for the other sex chromosome presence for conceptus to be compatible with life. In cases of structural chromosomal aberrations or hidden mosaicism, conventional cytogenetic techniques can be ineffective and molecular investigation is indicated. Two hundred and fifty patients with Turner syndrome stigmata were studied and 36 who had female genitalia and had been cytogenetically diagnosed as having "pure" 45,X karyotype were selected after 100 metaphases were analyzed in order to exclude mosaicism and the presence of genomic Y-specific sequences (SRY, TSPY, and DAZ) was excluded by PCR. Genomic DNA was extracted from peripheral blood and screened by the human androgen receptor (HUMARA) assay. The HUMARA gene has a polymorphic CAG repeat and, in the presence of a second chromosome with a different HUMARA allele, a second band will be amplified by PCR. Additionally, the CAG repeats contain two methylation-sensitive HpaII enzyme restriction sites, which can be used to verify skewed inactivation. Twenty-five percent (9/36) of the cases showed a cryptic mosaicism involving a second X and approximately 14 percent (5/36), or 55 percent (5/9) of the patients with cryptic mosaicism, also presented skewed inactivation. The laboratory identification of the second X chromosome and its inactivation pattern are important for the clinical management (hormone replacement therapy, and inclusion in an oocyte donation program) and prognostic counseling of patients with Turner syndrome.

The P48T germline mutation and polymorphism in the CDKN2A gene of patients with melanoma

CDKN2A has been implicated as a melanoma susceptibility gene in some kindreds with a family history of this disease. Mutations in CDKN2A may produce an imbalance between functional p16ink4a and cyclin D causing abnormal cell growth. We searched for germline mutations in this gene in 22 patients with clinical criteria of hereditary cancer (early onset, presence of multiple primary melanoma or 1 or more first- or second-degree relatives affected) by secondary structural content prediction, a mutation scanning method that relies on the propensity for single-strand DNA to take on a three-dimensional structure that is highly sequence dependent, and sequencing the samples with alterations in the electrophoretic mobility. The prevalence of CDKN2A mutation in our study was 4.5 percent (1/22) and there was a correlation between family history and probability of mutation detection. We found the P48T mutation in 1 patient with 2 melanoma-affected relatives. The patient descends from Italian families and this mutation has been reported previously only in Italian families in two independent studies. This leads us to suggest the presence of a mutational "hotspot" within this gene or a founder mutation. We also detected a high prevalence (59.1 percent) of polymorphisms, mainly alleles 500 C/G (7/31.8 percent) or 540 C/T (6/27.3 percent), in the 3' untranslated region of exon 3. This result reinforces the idea that these rare polymorphic alleles have been significantly associated with the risk of developing melanoma.

Vulvovaginites: estudo das variaçoes de pH vaginal e microflora associada / Vulvovaginitis: vaginal pH changes and associated microflora

PURPOSE: To establish a correlation between pH vaginal and the microflora associated in carriers of vulvovaginites. METHODS: In the present study, the cytopathological examination and the vaginal flow in a group of 65 sexually active women had been carried through, 20 and 72 years, taken care of in the Laboratório de Citologia Clínica do Departamento de Análises Clínicas e Toxicológicas da Universidade Federal do Rio Grande do Norte, for determination of microorganisms in cervicovaginal sample and of pH in the vaginal flow. RESULTS: Associating pH vaginal with the presence of vulvovaginitis, it was evidenced that the Candida sp. occurred more frequently in pH 4.0, Trichomonas vaginalis in pH 6.0, Gardnerella vaginalis in pH 5.0, coconuts in pH 5.0, bacilli in pH 4.0 and cocos/bacilos in pH 6.0. It was observed that all the patients had presented at least one type of ethiological agent of vulvovaginiti and an associated microflora. CONCLUSION: The joint accomplishment of the cytological examinations and the determination of pH revealed important for directing the microflora associated with the vulvovaginiti, suggesting, of this form, that pH vaginal plays preponderant role how much to the presence of the infectious agents in the vaginal ecosystem.

Importância da correlação do exame citológico com a captura híbrida II no diagnóstico de lesoes intraepiteliais cervicais relacionadas ao HPV1 / Correlation of hybrid II capture cytologic exam in diagnosis of cervical lesions related to HPV

PURPOSE: The aim of the present study was confront the results of the cytological examination with hybrid capture II in the diagnosis of induced cervical intraepithelial lesion-HPV, correlating the cytological findings with biomoleculares. METHODS: The research was carried through in a group of 160 sexually active women who had espontaneamente looked its gynecologists for consultation of routine, having been submitted to the collection of cervicovaginal material for cytology and for examination of hybrid capture II in the Centro de Patologia Clínica and the Hospital e Maternidade Promater, in the city of the Natal-RN. RESULTS: The results had shown to relatively high numbers of positive cases for HPV using hybrid capture II (41.87%) and the cytology (23.75%). The agreement between the two studied methods relatively was raised (59.38%). It was evident also that the viruses with high oncogênico potential had presented found in the compatible cytology with Lesion of low risk (11.88%), followed of Lesion of high risk (NIC II and III); already the viruses with low oncogênico potential were more associates the Lesion of low risk (6.25%), followed of Lesion of high risk. CONCLUSION: The cytology, exactly with its limitations, is an important method in the detention of attributable patologias to the HPV, emphasizing that the molecular method comes to complement it and to consolidate the cytological findings.

Trisomy 13 mosaicism demonstrated only in skin fibroblasts in a patient presenting psychomotor retardation, pigmentary dysplasia and some dysmorphic features

Os autores descrevem uma criança do sexo feminino com atraso do desenvolvimento neuropsicomotor, manchas hipercrômicas na pele e algumas características dismórficas. O cariótipo em linfócitos de sangue periférico foi normal e a cultura de fibroblastos a partir de biópsia de pele revelou trissomia do cromossomo 13 em 50 por cento das metáfases analisadas. Enfatizam a importância do cariótipo de pele nas situaçöes clínicas em que o retardo psicomotor ou mental está associado com displasia pigmentar de pele e cromossomos normais nos linfócitos.