Genome-wide association analysis identifies 20 loci that influence adult height.

Adult height is a model polygenic trait, but there has been limited success in identifying the genes underlying its normal variation. To identify genetic variants influencing adult human height, we used genome-wide association data from 13,665 individuals and genotyped 39 variants in an additional 16,482 samples. We identified 20 variants associated with adult height (P < 5 x 10(-7), with 10 reaching P < 1 x 10(-10)). Combined, the 20 SNPs explain approximately 3% of height variation, with a approximately 5 cm difference between the 6.2% of people with 17 or fewer 'tall' alleles compared to the 5.5% with 27 or more 'tall' alleles. The loci we identified implicate genes in Hedgehog signaling (IHH, HHIP, PTCH1), extracellular matrix (EFEMP1, ADAMTSL3, ACAN) and cancer (CDK6, HMGA2, DLEU7) pathways, and provide new insights into human growth and developmental processes. Finally, our results provide insights into the genetic architecture of a classic quantitative trait.

Sequencing of simple sequence repeat anchored polymerase chain reaction amplification products of Biomphalaria glabrata

Simple sequence repeat anchored polymerase chain reaction amplification (SSR-PCR) is a genetic typing technique based on primers anchored at the 5' or 3' ends of microsatellites, at high primer annealing temperatures. This technique has already been used in studies of genetic variability of several organisms, using different primer designs. In order to conduct a detailed study of the SSR-PCR genomic targets, we cloned and sequenced 20 unique amplification products of two commonly used primers, CAA(CT)6 and (CA)8RY, using Biomphalaria glabrata genomic DNA as template. The sequences obtained were novel B. glabrata genomic sequences. It was observed that 15 clones contained microsatellites between priming sites. Out of 40 clones, seven contained complex sequence repetitions. One of the repeats that appeared in six of the amplified fragments generated a single band in Southern analysis, indicating that the sequence was not widespread in the genome. Most of the annealing sites for the CAA(CT)6 primer contained only the six repeats found within the primer sequence. In conclusion, SSR-PCR is a useful genotyping technique. However, the premise of the SSR-PCR technique, verified with the CAA(CT)6 primer, could not be supported since the amplification products did not result necessarily from microsatellite loci amplification.

Polymerase chain reaction and restriction fragment length polymorphism of cytocrome oxidase subunit I used for differentiation of Brazilian Biomphalaria species intermediate host of Schistosoma mansoni

The intermediate hosts of Schistosoma mansoni, in Brazil, Biomphalaria glabrata, B. tenagophila and B. straminea, were identified by restriction fragment length polymorphism analysis of the mitochondrial gene cytochrome oxidase I (COI). We performed digestions with two enzymes (AluI and RsaI), previously selected, based on sequences available in Genbank. The profiles obtained with RsaI showed to be the most informative once they were polymorphic patterns, corroborating with much morphological data. In addition, we performed COI digestion of B. straminea snails from Uruguay and Argentina.

Cloning and Molecular Characterization of the Schistosoma mansoni Genes RbAp48 and Histone H4

The human nuclear protein RbAp48 is a member of the tryptophan/aspartate (WD) repeat family, which binds to the retinoblastoma (Rb) protein. It also corresponds to the smallest subunit of the chromatin assembly factor and is able to bind to the helix 1 of histone H4, taking it to the DNA in replication. A cDNA homologous to the human gene RbAp48 was isolated from a Schistosoma mansoni adult worm library and named SmRbAp48. The full length sequence of SmRbAp48 cDNA is 1036 bp long, encoding a protein of 308 amino acids. The transcript of SmRbAp48 was detected in egg, cercariae and schistosomulum stages. The protein shows 84% similarity with the human RbAp48, possessing four WD repeats on its C-terminus. A hypothetical tridimensional structure for the SmRbAp48 C-terminal domain was constructed by computational molecular modeling using the b-subunit of the G protein as a model. To further verify a possible interaction between SmRbAp48 and S. mansoni histone H4, the histone H4 gene was amplified from adult worm genomic DNA using degenerated primers. The gene fragment of SmH4 is 294 bp long, encoding a protein of 98 amino acids which is 100% identical to histone H4 from Drosophila melanogaster.

Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil

The prevalence of Mycobacterium bovis and other mycobacterial species in livestock specimens and milk was evaluated. An emphasis was placed upon the distribution of these organisms in milk that is readily available to the public that was either untreated, pasteurized, or treated using ultra high temperature. Twenty-two pathologic specimens from livestock (bovine, swine and bubaline) in five Brazilian states and 128 bovine milk samples from retail markets in the State of São Paulo were examined for mycobacteria. Identification was made by classical biochemical tests, thin layer chromatography of mycolic acids and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Mycobacteria were isolated from 15 (68.2%) caseous lesions and from 23 (18%) milk samples. Eleven isolates were identified as M. bovis, and the remaining 27 nontuberculous mycobacterial isolates were represented by five species and six unidentified rapidly growing mycobacterial strains. The data demonstrate that animal products in Brazil are frequent reservoirs of mycobacteria and may pose a risk to the public.

IS6110 restriction fragment length polymorphism of Mycobacterium tuberculosis isolated from patients with pulmonary tuberculosis in Campinas, Brazil: evidence of intercontinental distribution of strains

Tuberculosis (TB) is a major concern in developing countries. In Brazil, few genotyping studies have been conducted to verify the number of IS6110 copies present in local prevalent strains of Mycobacterium tuberculosis, the distribution and clustering of strains. IS6110 DNA fingerprinting was performed on a sample of M. tuberculosis isolates from patients with AFB smear-positive pulmonary TB, at a hospital in Brazil. The IS6110 profiles were analyzed and compared to a M. tuberculosis database of the Houston Tuberculosis Initiative, Houston, US. Seventy-six fingerprints were obtained from 98 patients. All M. tuberculosis strains had an IS6110 copy number between 5-21 allowing for differentiation of the isolates. Human immunodeficiency virus infection was confirmed in nearly half the patients of whom data was available. Fifty-eight strains had unique patterns, while 17 strains were grouped in 7 clusters (2 to 6 strains). When compared to the HTI database, 6 strains matched isolates from El Paso, Ciudad de Juarez, Houston, and New York. Recently acquired infections were documented in 19% of cases. The community transmission of infection is intense, since some clustered strains were recovered during the four-year study period. The intercontinental dissemination of M. tuberculosis strains is suspected by demonstration of identical fingerprints in a distant country.

Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by polymerase chain reaction- restriction fragment length polymorphism

Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2) and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.

Bowel cancer screening: the repeat test (English and 10 translations)

Format: 6 page A5 leaflet Target group: Participants in the Northern Ireland Bowel cancer screening programme who require a second test. Description: This leaflet provides step by step instructions on using the Faecal Immunochemical Test (FIT) for bowel cancer screening.

Dominant character of the molecular marker of a Biomphalaria tenagophila strain (Mollusca: Planorbidae) resistant to Schistosoma mansoni

Biomphalaria tenagophila population from Taim (state of Rio Grande do Sul, Brazil) is totally resistant toSchistosoma mansoni, and presents a molecular marker of 350 bp by polymerase chain reaction and restriction fragment length polymorphism of the entire rDNA internal transcriber spacer. The scope of this work was to determine the heritage pattern of this marker. A series of cross-breedings between B. tenagophila from Taim (resistant) and B. tenagophila from Joinville, state of Santa Catarina (susceptible) was carried out, and their descendants F1 and F2 were submitted to this technique. It was possible to demonstrate that the specific fragment from Taim is endowed with dominant character, since the obtained segregation was typically mendelian.

The fine-scale architecture of structural variants in 17 mouse genomes.

BACKGROUND: Accurate catalogs of structural variants (SVs) in mammalian genomes are necessary to elucidate the potential mechanisms that drive SV formation and to assess their functional impact. Next generation sequencing methods for SV detection are an advance on array-based methods, but are almost exclusively limited to four basic types: deletions, insertions, inversions and copy number gains. RESULTS: By visual inspection of 100 Mbp of genome to which next generation sequence data from 17 inbred mouse strains had been aligned, we identify and interpret 21 paired-end mapping patterns, which we validate by PCR. These paired-end mapping patterns reveal a greater diversity and complexity in SVs than previously recognized. In addition, Sanger-based sequence analysis of 4,176 breakpoints at 261 SV sites reveal additional complexity at approximately a quarter of structural variants analyzed. We find micro-deletions and micro-insertions at SV breakpoints, ranging from 1 to 107 bp, and SNPs that extend breakpoint micro-homology and may catalyze SV formation. CONCLUSIONS: An integrative approach using experimental analyses to train computational SV calling is essential for the accurate resolution of the architecture of SVs. We find considerable complexity in SV formation; about a quarter of SVs in the mouse are composed of a complex mixture of deletion, insertion, inversion and copy number gain. Computational methods can be adapted to identify most paired-end mapping patterns.

Analysis of the first and second internal transcribed spacer sequences of the ribosomal DNA in Biomphalaria tenagophila complex (Mollusca: Planorbidae)

The first and second internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA of Biomphalaria tenagophila complex (B. tenagophila, B. occidentalis, and B. t. guaibensis) were sequenced and compared. The alignment lengths of these regions were about 655 bp and 481 bp, respectively. Phylogenetic relationships among the Biomphalaria species were inferred by Maximum Parsimony and Neighbor-joining methods. The phylogenetic trees produced, in most of the cases, were in accordance with morphological systematics and other molecular data previously obtained by polymerase chain reaction and restriction fragment length polymorphism analysis. The present results provide support for the proposal that B. tenagophila represents a complex comprising B. tenagophila, B. occidentalis and B. t. guaibensis.

Colposcopic evaluation after a repeat atypical squamous cells of undetermined significance (ASCUS) smear.

OBJECTIVES: Management of patients with atypical squamous cells of undetermined significance (ASCUS) remains controversial. We chose to repeat the Pap smear after four months. If ASCUS persisted in this second test, the patient was advised to undergo colposcopy. Our objective is to determine the clinical significance and the prediction of neoplasia among these patients through a colposcopic examination. METHODS: Of 29,827 patients who had a Pap smear, ASCUS were detected in 1387 (5%) and persisted in the repeat smear of 225 (16%). Colposcopy and an additional Pap smear were performed on 186 patients. RESULTS: Out of 186 colposcopic evaluations, 91 (49%) were normal and the patients had a negative Pap smear. Colposcopy was abnormal in 95/186 patients (51%) (Table 1). Histology of the directed biopsies revealed 38 (21%) low-grade squamous intraepithelial lesions (LSIL) and 17 (9%) high-grade squamous intraepithelial lesions (HSIL). Forty patients (21%) with normal biopsies had ASCUS for the third time in the Pap smear. CONCLUSIONS: Colposcopic evaluation after a repeated Pap smear with ASCUS is an appropriate cost-effective management. Finding 30% of LSIL or HSIL justifies this additional investigation.

The use of the polymerase chain reaction and restriction fragment length polymorphism technique associated with the classical morphology for characterization of Lymnaea columella, L. viatrix, and L. diaphana (Mollusca: Lymnaeidae)

The specific identification of Lymnaeid snails is based on a comparison of morphological characters of the shell, radula, renal and reproductive organs. However, the identification is complicated by dissection process, intra and interspecific similarity and variability of morphological characters. In the present study, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques targeted to the first and second internal transcribed spacers (ITS1 and ITS2) rDNA and to the mitochondrial 16S ribosomal gene (16S rDNAmt) were used to differentiate the species Lymnaea columella, L. viatrix, and L. diaphana from some localities of Brazil, Argentina, and Uruguay as well as to verify whether the molecular results corroborates the classical morphological method.PCR-RFLP analysis of the ITS1, ITS2, and 16S using 12 restriction enzymes revealed characteristic patterns for L. columella and L. diaphana which were concordant with the classical morphology. On the other hand, for L. viatrix populations a number of 1 to 6 profiles were generated while morphology provided the species pattern results.

Mycobacterium tuberculosis complex differentiation using gyrB-restriction fragment length polymorphism analysis

Mycobacterium tuberculosis complex (MTBC) members are causative agents of human and animal tuberculosis. Differentiation of MTBC members is required for appropriate treatment of individual patients and for epidemiological purposes. Strains from six MTBC species - M. tuberculosis, M. bovis subsp. bovis, M. bovis BCG, M. africanum, M. pinnipedii, and "M. canetti" - were studied using gyrB-restriction fragment length polymorphism (gyrB-RFLP) analysis. A table was elaborated, based on observed restriction patterns and published gyrB sequences. To evaluate applicability of gyrB-RFLP at Instituto Adolfo Lutz, São Paulo, Mycobacterial Reference Laboratory, 311 MTBC clinical isolates, previously identified using traditional methods as M. tuberculosis (306), M. bovis (3), and M. bovis BCG (2), were analyzed by gyrB-RFLP. All isolates were correctly identified by the molecular method, but no distinction between M. bovis and M. bovis BCG was obtained. Differentiation of M. tuberculosis and M. bovis is of utmost importance, because they require different treatment schedules. In conclusion, gyrB-RFLP is accurate and easy-to-perform, with potential to reduce time needed for conventional differentiation methods. However, application for epidemiological studies remains limited, because it cannot differentiate M. tuberculosis from M. africanum subtype II, and "M. canetti", M. africanum subtype I from M. pinnipedii, and. M. bovis from M. bovis BCG.