Unexpected genetic heterogeneity in a large consanguineous Brazilian pedigree presenting deafness

Nonsyndromic autosomal recessive deafness accounts for 80% of hereditary deafness. To date, 52 loci responsible for autosomal recessive deafness have been mapped and 24 genes identified. Here, we report a large inbred Brazilian pedigree with 26 subjects affected by prelingual deafness. Given the extensive consanguinity found in this pedigree, the most probable pattern of inheritance is autosomal recessive. However, our linkage and mutational analysis revealed, instead of an expected homozygous mutation in a single gene, two different mutant alleles and a possible third undetected mutant allele in the MYO15A gene (DFNB3 locus), as well as evidence for other causes for deafness in the same pedigree. Among the 26 affected subjects, 15 were homozygous for the novel c.10573delA mutation in the MYO15A gene, 5 were compound heterozygous for the mutation c.10573delA and the novel deletion c.9957_9960delTGAC and one inherited only a single c.10573delA mutant allele, while the other one could not be identified. Given the extensive consanguinity of the pedigree, there might be at least one more deafness locus segregating to explain the condition in some of the subjects whose deafness is not clearly associated with MYO15A mutations, although overlooked environmental causes could not be ruled out. Our findings illustrate a high level of etiological heterogeneity for deafness in the family and highlight some of the pitfalls of genetic analysis of large genes in extended pedigrees, when homozygosity for a single mutant allele is expected.

The effect of a single episode of antimicrobial photodynamic therapy in the treatment of experimental periodontitis. Microbiological profile and cytokine pattern in the dog mandible

The purpose of this study was to evaluate the effect of a single application of antimicrobial photodynamic therapy (aPDT) on microbiological profile and cytokine pattern in dogs. Periodontal disease was induced by placing 3.0 silk ligatures around the mandibular pre-molars bilaterally during 8 weeks. The dogs were randomly treated with aPDT using a dye/laser system, scaling and root planning (SRP), or with the association of treatments (SRP + aPDT). Plaque samples were collected at baseline, 1, 3, and 4 weeks, and the mean counts of 40 species were determined using DNA-DNA hybridization. Gingival biopsies were removed and the expression of tumor necrosis factor alpha (TNF-alpha), receptor activator of NF-kB ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase (MMP-1), interleukin (IL) 6, IL-10 and total bacterial load by analysis of 16 S rRNA gene were evaluated through real-time PCR. The results shows that the levels of the majority of the species were reduced 1 week post-therapy for all treatments, however, an increase in counts of Prevotella intermedia (p = 0.00), Prevotella. nigrescens (p = 0.00) and Tannerella forsythia (p = 0.00) was observed for aPDT and SRP + aPDT. After 4 weeks, a regrowth of Porphyromonas gingivalis (p = 0.00) and Treponema denticola (p = 0.00), was observed for all treatments. Also, a strikingly reduction of counts on counts of Aggregatibacter actinomycetemcomitans was observed for the aPDT (p = 0.00). For the cytokine pattern, the results were similar for all treatments, and a reduction in the expression of cytokines and bacterial load was observed throughout the study. Our results suggest that SRP, aPDT in a single application, and SRP + aPDT affects different bacterial species and have similar effects on the expression of cytokines evaluated during the treatment of ligature-induced periodontitis.

Association of genetic variants in the adiponectin encoding gene (ADIPOQ) with type 2 diabetes in Japanese Brazilians

Aim: The objective of this study is to assess the contribution of ADIPOQ variants to type 2 diabetes in Japanese Brazilians. Methods: We genotyped 200 patients with diabetes mellitus (100 male and 100 female, aged 55.0 years [47.5-64.0 years]) and 200 control subjects with normal glucose tolerant (NGT) (72 male and 128 female, aged 52.0 years [43.5-64.5 years]). Results: Whereas each polymorphism studied (T45G, G276T, and A349G) was not significantly associated with type 2 diabetes mellitus, the haplotype GGA was overrepresented in our diabetic population (9.3% against 3.1% in NGT individuals, P=.0003). Also, this haplotype was associated with decreased levels of adiponectin. We also identified three mutations in exon 3: I164T, R221S, and H241P, but, owing to the low frequencies of them, associations with type 2 diabetes could not be evaluated. The subjects carrying the R221S mutation had plasma adiponectin levels lower than those without the mutation (2.10 mu g/ml [1.35-2.55 mu g/ml] vs. 6.68 mu g/ml [3.90-11.23 mu g/ml], P=.015). Similarly, the I164T mutation carriers had mean plasma adiponectin levels lower than those noncarriers (3.73 mu g/ml [3.10-4.35 mu g/ml] vs. 6.68 mu g/ml [3.90-11.23 mu g/ml]), but this difference was not significant (P=.17). Conclusions: We identified in the ADIPOQ gene a risk haplotype for type 2 diabetes in the Japanese Brazilian population. (C) 2010 Elsevier Inc. All rights reserved.

Development of new nuclear markers and characterization of single nucleotide polymorphisms in kelp gull (Larus dominicanus)

The present study seeks to develop nuclear markers for the kelp gull (Larus dominicanus). We hereby report the characterization of 12 independent nuclear introns, where 104 single nucleotide polymorphisms (SNPs) in 8138 sequenced base pairs were observed. These SNP markers are the first to be designed for genotyping a gull species. The markers will provide useful tools for understanding which processes act or acted upon kelp gulls to cause their low genetic variability in mitochondrial DNA. In addition, these markers open a new opportunity for population genetic and evolutionary studies in the Laridae group.

CHANGES OF GENE EXPRESSION IN ELECTRICALLY STIMULATED AND CONTRALATERAL RAT SOLEUS MUSCLES

In this study we investigate the effect of a single session of high-intensity contractions on expression of pleiotropic genes and, in particular, those genes associated with metabolism in soleus muscle from electrically stimulated (ES) and contralateral (CL) limbs. The right limbs of male Wistar rats were submitted to contractions by 200-ms trains of electrical stimulation at 100-Hz frequency with pulses of 0.1 ms (voltage 24 3 V) delivered each second for 1 hour. Soleus muscles were isolated 1 hour after contraction, and gene expression was analyzed by a macroarray technique (Atlas Toxicology 1.2 Array; Clontech Laboratories). Electrical stimulation increased expression in 92 genes (16% of the genes present in the membrane). Sixty-six genes were upregulated in both ES and CL soleus muscles, and expression of 26 genes was upregulated in the ES muscle only. The most altered genes were those related to stress response and metabolism. Electrical stimulation also raised expression of transcription factors, translation and posttranslational modification of proteins, ribosomal proteins, and intracellular transducers/effectors/modulators. The results indicate that a single session of electrical stimulation upregulated expression of genes related to metabolism and oxidative stress in soleus muscle from both ES and CL limbs. These findings may indicate an association with tissue hypertrophy and metabolic adaptations induced by physical exercise training not only in the ES but also in the CL non-stimulated muscle, suggesting a cross-education phenomenon. Muscle Nerve 40: 838-846, 2009

An interleukin-1 beta (IL-1 beta) single-nucleotide polymorphism at position 3954 and red complex periodontopathogens independently and additively modulate the levels of IL-1 beta in diseased periodontal tissues

Inflammatory cytokines such as interieukin-1 beta (IL-1 beta) are involved in the pathogenesis of periodontal diseases. A high individual variation in the levels of IL-10 mRNA has been verified, which is possibly determined by genetic polymorphisms and/or by the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans. In this study, we investigated the role of an IL-10 promoter single-nucleotide polymorphism at position 3954 [IL-1 beta(3954) SNP] and the presence of the periodontopathogens in the determination of the IL-1 beta levels in the periodontal tissues of nonsmoking chronic periodontitis (CP) patients (n = 117) and control (C) subjects in = 175) and the possible correlations with the clinical parameters of the disease. IL-1 beta(3954) SNP was investigated by restriction fragment length polymorphism, while the IL-1 beta levels and the presence of the periodontopathogens were determined by real-time PCR. Similar frequencies of IL-1 beta(3954) SNP were found in the C and CP groups, in spite of a trend toward a higher incidence of T alleles in the CP group. The IL-1 beta (3954) SNP CT and TT genotypes, as well as P. gingivalis, T. forsythia, and T. denticola, were associated with higher IL-1 beta levels and with higher values of the clinical parameters of disease severity. Concomitant analyses demonstrate that IL-1 beta(3954) and the red complex periodontopathogens were found to independently and additively modulate the levels of IL-1 beta in periodontal tissues. Similarly, the concurrent presence of both factors was associated with increased scores of disease severity. IL-1 beta(3954) genotypes and red complex periodontopathogens, individually and additively, modulate the levels of IL-1 beta in the diseased tissues of nonsmoking CP patients and, consequently, are potentially involved in the determination of the disease outcome.

Tumor necrosis factor-alpha-308G/A single nucleotide polymorphism and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-alpha in diseased periodontal tissues

Background and Objective: Inflammatory cytokines such as tumor necrosis factor-alpha are involved in the pathogenesis of periodontal diseases. A high between-subject variation in the level of tumor necrosis factor-alpha mRNA has been verified, which may be a result of genetic polymorphisms and/or the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola (called the red complex) and Aggregatibacter actinomycetemcomitans. In this study, we investigated the effect of the tumor necrosis factor-alpha (TNFA) -308G/A gene polymorphism and of periodontopathogens on the tumor necrosis factor-alpha levels in the periodontal tissues of nonsmoking patients with chronic periodontitis (n = 127) and in control subjects (n = 177). Material and Methods: The TNFA-308G/A single nucleotide polymorphism was investigated using polymerase chain reaction-restriction fragment length polymorphism analysis, whereas the tumor necrosis factor-alpha levels and the periodontopathogen load were determined using real-time polymerase chain reaction. Results: No statistically significant differences were found in the frequency of the TNFA-308 single nucleotide polymorphism in control and chronic periodontitis groups, in spite of the higher frequency of the A allele in the chronic periodontitis group. The concomitant analyses of genotypes and periodontopathogens demonstrated that TNFA-308 GA/AA genotypes and the red-complex periodontopathogens were independently associated with increased levels of tumor necrosis factor-alpha in periodontal tissues, and no additive effect was seen when both factors were present. P. gingivalis, T. forsythia and T. denticola counts were positively correlated with the level of tumor necrosis factor-alpha. TNFA-308 genotypes were not associated with the periodontopathogen detection odds or with the bacterial load. Conclusion: Our results demonstrate that the TNFA-308 A allele and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-alpha in diseased tissues of nonsmoking chronic periodontitis patients and consequently are potentially involved in determining the disease outcome.

Genetic and Functional Evidence Implicating DLL1 as the Gene That Influences Susceptibility to Visceral Leishmaniasis at Chromosome 6q27

Background. Visceral leishmaniasis (VL) is caused by Leishmania donovani and Leishmania infantum chagasi. Genome-wide linkage studies from Sudan and Brazil identified a putative susceptibility locus on chromosome 6q27. Methods. Twenty-two single-nucleotide polymorphisms (SNPs) at genes PHF10, C6orf70, DLL1, FAM120B, PSMB1, and TBP were genotyped in 193 VL cases from 85 Sudanese families, and 8 SNPs at genes PHF10, C6orf70, DLL1, PSMB1, and TBP were genotyped in 194 VL cases from 80 Brazilian families. Family-based association, haplotype, and linkage disequilibrium analyses were performed. Multispecies comparative sequence analysis was used to identify conserved noncoding sequences carrying putative regulatory elements. Quantitative reverse-transcription polymerase chain reaction measured expression of candidate genes in splenic aspirates from Indian patients with VL compared with that in the control spleen sample. Results. Positive associations were observed at PHF10, C6orf70, DLL1, PSMB1, and TBP in Sudan, but only at DLL1 in Brazil (combined P = 3 x 10(-4) at DLL1 across Sudan and Brazil). No functional coding region variants were observed in resequencing of 22 Sudanese VL cases. DLL1 expression was significantly (P = 2 x 10(-7)) reduced (mean fold change, 3.5 [SEM, 0.7]) in splenic aspirates from patients with VL, whereas other 6q27 genes showed higher levels (1.27 x 10(-6) < P < .01) than did the control spleen sample. A cluster of conserved noncoding sequences with putative regulatory variants was identified in the distal promoter of DLL1. Conclusions. DLL1, which encodes Delta-like 1, the ligand for Notch3, is strongly implicated as the chromosome 6q27 VL susceptibility gene.

Trypanosome Prereplication Machinery Contains a Single Functional Orc1/Cdc6 Protein, Which Is Typical of Archaea

In unicellular eukaryotes, such as Saccharomyces cerevisiae, and in multicellular organisms, the replication origin is recognized by the heterohexamer origin recognition complex (ORC) containing six proteins, Orc1 to Orc6, while in members of the domain Archaea, the replication origin is recognized by just one protein, Orc1/Cdc6; the sequence of Orc1/Cdc6 is highly related to those of Orc1 and Cdc6. Similar to Archaea, trypanosomatid genomes contain only one gene encoding a protein named Orc1. Since trypanosome Orc1 is also homologous to Cdc6, in this study we named the Orc1 protein from trypanosomes Orc1/Cdc6. Here we show that the recombinant Orc1/Cdc6 from Trypanosoma cruzi (TcOrc1/Cdc6) and from Trypanosoma brucei (TbOrc1/Cdc6) present ATPase activity, typical of prereplication machinery components. Also, TcOrc1/Cdc6 and TbOrc1/Cdc6 replaced yeast Cdc6 but not Orc1 in a phenotypic complementation assay. The induction of Orc1/Cdc6 silencing by RNA interference in T. brucei resulted in enucleated cells, strongly suggesting the involvement of Orc1/Cdc6 in DNA replication. Orc1/Cdc6 is expressed during the entire cell cycle in the nuclei of trypanosomes, remaining associated with chromatin in all stages of the cell cycle. These results allowed us to conclude that Orc1/Cdc6 is indeed a member of the trypanosome prereplication machinery and point out that trypanosomes carry a prereplication machinery that is less complex than other eukaryotes and closer to archaea.

The South American Plasmodium falciparum var gene repertoire is limited, highly shared and possibly lacks several antigenic types

The Plasmodium falciparum var gene family encodes large variant antigens, which are important virulence factors, and also targets of the humoral host response. The frequently observed mild outcomes of falciparum malaria in many places of the Amazon area prompted us to ask whether a globally restricted variant (var) gene repertoire is present in currently circulating and older isolates of this area. By exhaustive analysis of var gene tags from 89 isolates and clones taken during many years from all over the Brazilian Amazon, we estimate that there are probably no more than 350-430 distinct sequence types, less than for any similar sized area studied so far. Detailed analysis of the var tags from genetically distinct clones obtained from single isolates revealed restricted and redundant repertoires suggesting either a low incidence of infective bites or restricted variant gene diversity in inoculated parasites. Additionally, we found a structuring of var gene repertoires observed as a higher pairwise typing sharing in isolates from the same microregion compared to isolates from different regions. Fine analysis of translated var tags revealed that certain Distinct Sequence Identifiers (DSIDs) were differently represented in Brazilian/South American isolates when compared to datasets from other continents. By global alignment of worldwide var DBL alpha sequences and sorting in groups with more than 76% identity, 125 clusters were formed and more than half of all genes were found in nine clusters with 50 or more sequences. While Brazilian/South American sequences were represented only in 64 groups, African sequences were found in the majority of clusters. DSID type 1 related sequences accumulated almost completely in one single cluster, indicating that limited recombination occurs in these specific var gene types. These data demonstrate the so far highest pairwise type sharing values for the var gene family in isolates from all over an entire subcontinent. The apparent lack of specific sequences types suggests that the P. falciparum transmission dynamics in the whole Amazon are probably different from any other endemic region studied and possibly interfere with the parasite`s ability to efficiently diversify its variant gene repertoires. (C) 2010 Elsevier B.V. All rights reserved.

Intraspecific sequence variation in 16S rRNA gene of Ureaplasma diversum isolates

Ureaplasma diversum infection in bulls may result in seminal vesiculitis, balanoposthitis and alterations in spermatozoids. In cows, it can cause placentitis, fetal alveolitis, abortion and the birth of weak calves. U. diversum ATCC 49782 (serogroups A), ATCC 49783 (serogroup C) and 34 field isolates were used for this study. These microorganisms were submitted to Polymerase Chain Reaction for 16S gene sequence determination using Tact High Fidelity and the products were purified and bi-directionally sequenced. Using the sequence obtained, a fragment containing four hypervariable regions was selected and nucleotide polymorphisms were identified based on their position within the 16S rRNA gene. Forty-four single nucleotide polymorphisms (SNP) were detected. The genotypic variability of the 16S rRNA gene of U. diversum isolates shows that the taxonomy classification of these organisms is likely much more complex than previously described and that 16S rRNA gene sequencing may be used to suggest an epidemiologic pattern of different origin strains. (c) 2011 Elsevier B.V. All rights reserved.

Screening of ARHSP-TCC Patients Expands the Spectrum of SPG11 Mutations and Includes a Large Scale Gene Deletion

Autosomal recessive spastic paraplegia with thinning of corpus callosum (ARHSP-TCC) is a complex form of HSP initially described in Japan but subsequently reported to have a worldwide distribution with a particular high frequency in multiple families from the Mediterranean basin. We recently showed that ARHSP-TCC is commonly associated with mutations in SPG11/KIAA1840 on chromosome 15q. We have now screened a collection of new patients mainly originating from Italy and Brazil, in order to further ascertain the spectrum of mutations in SPG11, enlarge the ethnic origin of SPG11 patients, determine the relative frequency at the level of single Countries (i.e., Italy), and establish whether there is one or more common mutation. In 25 index cases we identified 32 mutations; 22 are novel, including 9 nonsense, 3 small deletions, 4 insertions, 1 in/del, 1 small duplication, 1 missense, 2 splice-site, and for the first time a large genomic rearrangement. This brings the total number of SPG11 mutated patients in the SPATAX collection to 111 cases in 44 families and in 17 isolated cases, from 16 Countries, all assessed using homogeneous clinical criteria. While expanding the spectrum of mutations in SPG11, this larger series also corroborated the notion that even within apparently homogeneous population a molecular diagnosis cannot be achieved without full gene sequencing. (C) 2008 Wiley-Liss, Inc.

Symmetry in biology: from genetic code to stochastic gene regulation

Mathematical models, as instruments for understanding the workings of nature, are a traditional tool of physics, but they also play an ever increasing role in biology - in the description of fundamental processes as well as that of complex systems. In this review, the authors discuss two examples of the application of group theoretical methods, which constitute the mathematical discipline for a quantitative description of the idea of symmetry, to genetics. The first one appears, in the form of a pseudo-orthogonal (Lorentz like) symmetry, in the stochastic modelling of what may be regarded as the simplest possible example of a genetic network and, hopefully, a building block for more complicated ones: a single self-interacting or externally regulated gene with only two possible states: ` on` and ` off`. The second is the algebraic approach to the evolution of the genetic code, according to which the current code results from a dynamical symmetry breaking process, starting out from an initial state of complete symmetry and ending in the presently observed final state of low symmetry. In both cases, symmetry plays a decisive role: in the first, it is a characteristic feature of the dynamics of the gene switch and its decay to equilibrium, whereas in the second, it provides the guidelines for the evolution of the coding rules.