Phenotypic and mutational spectrum of ROR2-related Robinow syndrome.

Robinow syndrome is characterized by a triad of craniofacial dysmorphisms, disproportionate-limb short stature, and genital hypoplasia. A significant degree of phenotypic variability seems to correlate with different genes/loci. Disturbances of the noncanonical WNT-pathway have been identified as the main cause of the syndrome. Biallelic variants in ROR2 cause an autosomal recessive form of the syndrome with distinctive skeletal findings. Twenty-two patients with a clinical diagnosis of autosomal recessive Robinow syndrome were screened for variants in ROR2 using multiple molecular approaches. We identified 25 putatively pathogenic ROR2 variants, 16 novel, including single nucleotide variants and exonic deletions. Detailed phenotypic analyses revealed that all subjects presented with a prominent forehead, hypertelorism, short nose, abnormality of the nasal tip, brachydactyly, mesomelic limb shortening, short stature, and genital hypoplasia in male patients. A total of 19 clinical features were present in more than 75% of the subjects, thus pointing to an overall uniformity of the phenotype. Disease-causing variants in ROR2, contribute to a clinically recognizable autosomal recessive trait phenotype with multiple skeletal defects. A comprehensive quantitative clinical evaluation of this cohort delineated the phenotypic spectrum of ROR2-related Robinow syndrome. The identification of exonic deletion variant alleles further supports the contention of a loss-of-function mechanism in the etiology of the syndrome.

Use of Targeted Exome Sequencing for Molecular Diagnosis of Skeletal Disorders.

Genetic disorders of the skeleton comprise a large group of more than 450 clinically distinct and genetically heterogeneous diseases associated with mutations in more than 300 genes. Achieving a definitive diagnosis is complicated due to the genetic heterogeneity of these disorders, their individual rarity and their diverse radiographic presentations. We used targeted exome sequencing and designed a 1.4 Mb panel for simultaneous testing of more than 4,800 exons in 309 genes involved in skeletal disorders. DNA from 69 individuals from 66 families with a known or suspected clinical diagnosis of a skeletal disorder was analyzed. Of 36 cases with a specific clinical hypothesis with a known genetic basis, mutations were identified for eight cases (22%). Of 20 cases with a suspected skeletal disorder but without a specific diagnosis, four causative mutations were identified. Also included were 11 cases with a specific skeletal disorder but for which there was at the time no known associated gene. For these cases, one mutation was identified in a known skeletal disease genes, and re-evaluation of the clinical phenotype in this case changed the diagnoses from osteodysplasia syndrome to Apert syndrome. These results suggest that the NGS panel provides a fast, accurate and cost-effective molecular diagnostic tool for identifying mutations in a highly genetically heterogeneous set of disorders such as genetic skeletal disorders. The data also stress the importance of a thorough clinical evaluation before DNA sequencing. The strategy should be applicable to other groups of disorders in which the molecular basis is largely known.

Population analysis of pharmacogenetic polymorphisms related to acute lymphoblastic leukemia drug treatment.

This study aimed to evaluate in the Brazilian population, the genotypes and population frequencies of pharmacogenetic polymorphisms involved in the response to drugs used in treatment of acute lymphoblastic leukemia (ALL), and to compare the data with data from the HapMap populations. There was significant differentiation between most population pairs, but few associations between genetic ancestry and SNPs in the Brazilian population were observed. AMOVA analysis comparing the Brazilian population to all other populations retrieved from HapMap pointed to a genetic proximity with the European population. These associations point to preclusion of the use of genetic ancestry as a proxy for predicting drug response. In this way, any study aiming to correlate genotype with drug response in the Brazilian population should be based on pharmacogenetic SNP genotypes.

Polymorphism analysis of the CTLA-4 gene in paracoccidioidomycosis patients.

The CTLA-4 protein is expressed in activated T cells and plays an essential role in the immune response through its regulatory effect on T cell activation. Polymorphisms of the CTLA-4 gene have been correlated with autoimmune, neoplastic and infectious illnesses. This work aimed to verify possible associations between single nucleotide polymorphisms (SNPs) in CTLA-4, -318C/T in the promoter and +49A/G in exon 1 and paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis. For this purpose, 66 chronic form PCM patients and 76 healthy controls had their allele, genotype and haplotype frequencies determined. The genetic admixture structure of the patients and controls was evaluated to eliminate ancestral bias. The comparison of frequencies indicated no significant differences between patients and controls that could link the SNPs to PCM. Groups were admixture matched with no difference observed in population ancestry inference, indicating that the absence of association between CTLA-4 polymorphisms and PCM could not be attributed to ancestral bias. This study showed that there was no association between the CTLA-4 SNPs -318 and +49 and the resistance or susceptibility to PCM.

Polymorphism analysis of the CTLA-4 gene in paracoccidioidomycosis patients

The CTLA-4 protein is expressed in activated T cells and plays an essential role in the immune response through its regulatory effect on T cell activation. Polymorphisms of the CTLA-4 gene have been correlated with autoimmune, neoplastic and infectious illnesses. This work aimed to verify possible associations between single nucleotide polymorphisms (SNPs) in CTLA-4, -318C/T in the promoter and +49A/G in exon 1 and paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis. For this purpose, 66 chronic form PCM patients and 76 healthy controls had their allele, genotype and haplotype frequencies determined. The genetic admixture structure of the patients and controls was evaluated to eliminate ancestral bias. The comparison of frequencies indicated no significant differences between patients and controls that could link the SNPs to PCM. Groups were admixture matched with no difference observed in population ancestry inference, indicating that the absence of association between CTLA-4 polymorphisms and PCM could not be attributed to ancestral bias. This study showed that there was no association between the CTLA-4 SNPs -318 and +49 and the resistance or susceptibility to PCM.

Population analysis of vitamin D receptor polymorphisms and the role of genetic ancestry in an admixed population

The vitamin D receptor (VDR) is an essential protein related to bone metabolism. Some VDR alleles are differentially distributed among ethnic populations and display variable patterns of linkage disequilibrium (LD). In this study, 200 unrelated Brazilians were genotyped using 21 VDR single nucleotide polymorphisms (SNPs) and 28 ancestry informative markers. The patterns of LD and haplotype distribution were compared among Brazilian and the HapMap populations of African (YRI), European (CEU) and Asian (JPT+CHB) origins. Conditional regression and haplotype-specific analysis were performed using estimates of individual genetic ancestry in Brazilians as a quantitative trait. Similar patterns of LD were observed in the 5' and 3' gene regions. However, the frequency distribution of haplotype blocks varied among populations. Conditional regression analysis identified haplotypes associated with European and Amerindian ancestry, but not with the proportion of African ancestry. Individual ancestry estimates were associated with VDR haplotypes. These findings reinforce the need to correct for population stratification when performing genetic association studies in admixed populations.

Microsatellite instability in endometrial polyps.

OBJECTIVE(S): To investigate the prevalence of microsatellite instability (MSI) in endometrial polyps and to evaluate whether there are clinical and histopathological parameters associated with this kind of instability. STUDY DESIGN: Between September 2008 and April 2009, endometrial polyps were collected from 109 patients. MSI was evaluated using the NCI recommended markers BAT25, BAT26, D2S123, D5S346 and D17S250. Histopathological analysis was performed, and clinical information was obtained from patients' records. RESULT(S): MSI low was detected in 6.4% of the validated samples (7/109). Of the seven MSI that were detected, six were positive for instability at D17S250 and one at D5S346. There were no significant differences between polyps with or without MSI with regard to age, BMI, menarche, parity, miscarriage or menopause; however, MSI was more frequent in polyps with simple hyperplasia without atypia (3/20; 15%). Furthermore, patients with multiple polyps had a marginally but statistically insignificant increase in the frequency of MSI (p<0.07). CONCLUSION(S): This is the first prospective study of MSI in endometrial polyps using hysteroscopically obtained samples. In a population of 109 patients, MSI was infrequent in endometrial polyps. Although MSI appears to be more frequent in multiple polyps and polyps with simple hyperplasia without atypia, this was not statistically significant.

Comparative proteomical and metalloproteomical analyses of human plasma from patients with laryngeal cancer.

Laryngeal cancer is a significant disease worldwide, which presents an increasing incidence. Two contrasting ideas of the immune system role during cancer development are accepted: (1) it fights tumor cells, and (2) it aids tumor progression. Thus, there is no clear understanding about the immune response in laryngeal cancer. Furthermore, since tobacco is the main cause of laryngeal cancer and it contains various carcinogenic components, including metallic elements, these may play a role on cancer development. Plasmas of patients with laryngeal cancer and of healthy smokers were evaluated by 2D gel electrophoresis and mass spectrometry. Proteins were detected on every gel around pH 4.0-10.0 from molecular mass of 10-60 kDa. Few differences were found among cancer and control patients. However, three spots gathered between pI 7.3 and 7.6 with different molecular masses appeared exclusively in cancer profiles. From ten spots identified, six correspond to immune system components, including the three differential ones. The latter were observed only in cancer patients. The presence of several trace elements in the identified proteins was determined by inductively coupled plasma mass spectrometry, where chromium was increased in all proteins analyzed from patients with cancer. This study reinforces the importance of the immune response as target in the understanding and treatment of laryngeal cancer and the possibility that chromium is important in the carcinogenic progress.

Estudo comparativo da função do assoalho pélvico em mulheres continentes e incontinentes na pós menopausa / Comparative study of pelvic floor function in continent and incontinent postmenopausal women

CONTEXTUALIZAÇÃO: A incontinência urinária (IU) é de causa multifatorial, sendo atribuída, em parte, à fraqueza da musculatura do assoalho pélvico. Apesar de ser subestimada por muitas mulheres, a avaliação funcional do assoalho pélvico (AFA) pode contribuir para um correto diagnóstico e terapêutica adequada. OBJETIVOS: Comparar a função muscular do assoalho pélvico em mulheres continentes e incontinentes na pós menopausa como fator diagnóstico no tratamento da IU. MÉTODOS: A partir da investigação dos sintomas urinários, 153 mulheres (idade X=66,7±5,4) foram separadas em dois grupos (G1 incontinentes e G2 assintomáticas). Após análise dos critérios de inclusão, as mulheres foram submetidas à AFA por meio da palpação bidigital (classificação de Contreras Ortis, 1994) e à quantificação da pressão de contração perineal por meio do perineômetro (PERINA 996-2® QUARK). RESULTADOS: Observou-se prevalência de IU (54,9 por cento) na amostra estudada, sendo a incontinência urinária de esforço (IUE) (41,7 por cento) o tipo mais presente. Em relação aos sintomas urinários, como a frequência miccional diurna (p=0,004) e noturna (p=0,02), o grupo G1 apresentou um valor significativamente mais alto. A AFA mostrou resultados similares durante a palpação e o perineômetro, com diferenças significativas (p<0,001) entre os dois grupos. Utilizou-se estatística descritiva, teste t de Student para amostras independentes, medidas de prevalência e análise de variância (one-way ANOVA), seguida do post hoc de Bonferroni (p<0,05). O software Statistical Package for the Social Sciences (SPSS) versão 10,0 (SPSS, Chicago, IL) foi utilizado para realização de todas as análises. CONCLUSÕES: A palpação e o perineômetro se mostraram eficientes na avaliação da força e pressão de contração desse grupo muscular.

BACKGROUND: Urinary incontinence (UI) is multifactorial and attributed, in part, to weakness of the pelvic floor muscles. Despite being underestimated by many women, a functional pelvic floor assessment (FPA) may contribute to a correct diagnosis and appropriate treatment. OBJECTIVES: To compare the function of pelvic floor muscles in continent and incontinent postmenopausal women as a diagnostic factor in UI treatment. METHODS: Based on the investigation of urinary symptoms, 153 women (age X=66.7±5.4) were divided into two groups (G1-incontinent and G2-continent). After analysis of the inclusion criteria, the women were submitted to FPA by means of bidigital palpation according to Contreras Ortiz (1994) and quantification of perineal strength with a perineometer (PERINA 996-2 QUARK®). RESULTS: There was prevalence of UI (54.9 percent) in the sample, with stress urinary incontinence (41.7 percent) as the most common. Regarding urinary symptoms such as diurnal (p=0.004) and nocturnal (p=0.02) voiding frequency, G1 had a significantly higher value. The FPA found similar results via palpation and the perineometer, with significant differences (p<0.001) between the two groups. We used descriptive statistics, the Student t test for independent samples, measures of prevalence and one-way ANOVA, followed by Bonferroni's post-hoc test (p<0.05). The software Statistical Package for the Social Sciences (SPSS) version 10.0 (SPSS, Chicago, IL) was used to perform all tests. CONCLUSIONS: Palpation and the perineometer were efficient forms of assessing the force and pressure of the muscle contractions of this muscle group.

A linkage map for the B-genome of Arachis (Fabaceae) and its synteny to the A-genome.

BACKGROUND: Arachis hypogaea (peanut) is an important crop worldwide, being mostly used for edible oil production, direct consumption and animal feed. Cultivated peanut is an allotetraploid species with two different genome components, A and B. Genetic linkage maps can greatly assist molecular breeding and genomic studies. However, the development of linkage maps for A. hypogaea is difficult because it has very low levels of polymorphism. This can be overcome by the utilization of wild species of Arachis, which present the A- and B-genomes in the diploid state, and show high levels of genetic variability. RESULTS: In this work, we constructed a B-genome linkage map, which will complement the previously published map for the A-genome of Arachis, and produced an entire framework for the tetraploid genome. This map is based on an F2 population of 93 individuals obtained from the cross between the diploid A. ipaënsis (K30076) and the closely related A. magna (K30097), the former species being the most probable B genome donor to cultivated peanut. In spite of being classified as different species, the parents showed high crossability and relatively low polymorphism (22.3%), compared to other interspecific crosses. The map has 10 linkage groups, with 149 loci spanning a total map distance of 1,294 cM. The microsatellite markers utilized, developed for other Arachis species, showed high transferability (81.7%). Segregation distortion was 21.5%. This B-genome map was compared to the A-genome map using 51 common markers, revealing a high degree of synteny between both genomes. CONCLUSION: The development of genetic maps for Arachis diploid wild species with A- and B-genomes effectively provides a genetic map for the tetraploid cultivated peanut in two separate diploid components and is a significant advance towards the construction of a transferable reference map for Arachis. Additionally, we were able to identify affinities of some Arachis linkage groups with Medicago truncatula, which will allow the transfer of information from the nearly-complete genome sequences of this model legume to the peanut crop.

Frequency of the CCR5delta32 allele in Brazilians: a study in colorectal cancer and in HTLV-I infection

A observaçäo de que indivíduos homozigotos para uma deleçäo de 32 pares de base no gene que codifica para o receptor 5 de cc-quimiocinas apresentam um menor risco de contrair a infecçäo por HIV-1 levou à investigaçäo da freqüência deste polimorfismo em várias populaçöes mundiais. É importante investigar se o CCR5delta32 é um fator a ser considerado na epidemiologia do HIV em populaçöes individuais. Com estes pressupostos em mente nós estabelecemos a freqüência do CCR5delta32 em uma grande amostra (907 indivíduos näo-relacionados) da populaçäo urbana do sudeste brasileiro, estratificada da seguinte maneira: 322 indivíduos sadios, 354 pacientes com câncer colorretal e 229 doadores de sangue. Os três grupos apresentaram essencialmente a mesma freqüência alélica de CCR5delta32 e a comparaçäo par-a-par näo revelou diferenças significativas. Assim, os nossos resultados podem ser agrupados para fornecer uma estimativa confiável de 0,053 ñ 0,005 da freqüência alélica de CCR5delta32. Os doadores de sangue compreendiam 50 indivíduos soronegativos para HTLV-I, 115 indivíduos assintomáticos por ELISA mas com resultados indeterminados em Western blot, 49 indivíduos soropositivos para HTLV-I mas assintomáticos e 15 indivíduos soropositivos para HTLV-I sintomáticos com mielopatia. Foi observado um sugestivo gradiente decrescente da freqüência alélica de CCR5delta32 nestas categorias. Entretanto, quando aplicamos o teste exato de Fisher, näo emergiram diferenças significativas. Para uma melhor avaliaçäo da influência do alelo CCR5delta32 na probabilidade de infectar-se com HTLV-I ou de desenvolver doença clínica seräo necessários estudos com um maior número de doadores de sangue.

Screening fetal losses for monosomy X with a simple PCR-based procedure

Para a detecçäo molecular de monossomia X em perdas fetais espontâneas, nós exploramos uma estratégia baseada em perda e heterozigosidade, desenvolvendo um sistema multiplex fluorescente que permite a amplificaçäo simultânea de cinco microssatélites em uma regiäo com baixa recombinaçäo no cromossomo X. A análise foi entäo feita por densitometria a laser assistida por computador. Nenhum caso de homozigosidade em todos os cinco locos foi encontrado em 30 mulheres normais estudadas, nem em 37 mulheres cuja tipagem foi extraída do banco de dados do CEPH. Além disso, todos os casos de monossomia X previamente diagnosticadas por citogenética convencional apresentaram a prevista perda de heterozigosidade. Quando estudamos 19 casos de perdas fetais femininas do primeiro trimestre da gravidez, encontramos quatro amostras uniformemente homozigotas em todos os locos (21 por cento), de acordo com a proporçäo esperada de casos de monossomia X em perdas fetais do primeiro trimestre. Concluímos que o sistema multiplex que nós desenvolvemos apresenta alta diversidade e alta eficiência de amplificaçäo pela PCR e constitui um método simples e útil de triagem para monossomia X em abortamentos e natimortos.