Protective effect of interleukin-23A (IL23A) haplotype variants on type 1A diabetes mellitus in a Brazilian population.

The Interleukin 23 (IL-23) has a central role in autoimmunity. Allelic variants of p19 subunit of IL-23 (IL23A) and IL-23 receptor (IL23R) genes and increased IL-23 serum concentrations were associated with autoimmune diseases. We therefore searched for variants of IL23A and IL23R that could predispose to Type 1 diabetes (T1D). The coding regions and boundary intron sequences of IL23A were sequenced. Variants of IL23A and of IL23R were also genotyped. Pancreatic and extrapancreatic autoantibodies and IL-23 serum levels were determined. The cohort involved 370 patients with T1D and 351 healthy control subjects. We observed only one coding IL23A variant (rs11171806 G>A) out of the 6 described in databases. As the G alleles of rs11171806 and rs2066808 variants of IL23A gene were in strong linkage disequilibrium (D'=-0.825 for controls, p<2.0 × 10(-6) and D'=-0.902, p<2.0 × 10(-17) for patients), further analyses were performed with the haplotypes. The GG haplotype was more frequent in controls (16.7%) than in T1D patients (9.5%), conferring a protection to T1D (OR=0.53; pc=0.0003). No association was found between IL23A allelic variants with age at diagnosis of diabetes, C-peptide levels or frequency of autoantibodies. IL23R variants (rs10889677 and rs11209026) frequency and IL-23 serum concentrations were similar between groups. The GG haplotype of lL23A variants (rs11171806 and rs2066808) was protective against T1D. IL23R variants (rs11209026 and rs10889677) were not associated with T1D. IL-23 serum concentrations did not differ between groups.

The PTPN22 1858T allele but not variants in the proximal promoter region of IL-21 gene is associated with the susceptibility to type 1 diabetes and the presence of autoantibodies in a Brazilian cohort.

Interleukin (IL)-21 and protein tyrosine phosphatase non-receptor 22 (PTPN22) regulate lymphocyte function and have been implicated in the pathogenesis of autoimmune diabetes. We sequenced the proximal promoter of the IL-21 gene for the first time and analysed the PTPN22 1858T polymorphism in type 1A diabetes (T1AD) patients and healthy controls (HC). We correlated the frequencies of islet and extra-pancreatic autoantibodies with genotypes from both loci. The case series comprised 612 T1AD patients and 792 HC. Genotyping of PTPN22 C1858T was performed on 434 T1AD patients and 689 HC. The -448 to +83 base pairs (bp) region of the IL-21 gene was sequenced in 309 Brazilian T1AD and 189 HC subjects. We also evaluated human leucocyte antigen (HLA) DR3/DR4 alleles. The frequencies of glutamic acid decarboxylase (GAD65), tyrosine phosphatase-like protein (IA)-2, anti-nuclear antibody (ANA), thyroid peroxidase (TPO), thyroglobulin (TG), thyrotrophin receptor autoantibody (TRAb), anti-smooth muscle (ASM) and 21-hydroxylase (21-OH) autoantibodies were higher in T1AD patients than in HC. The PTPN22 1858T allele was associated with an increased risk for developing T1AD [odds ratio (OR) = 1·94; P < 0·001], particularly in patients of European ancestry, and with a higher frequency of GAD65 and TG autoantibodies. HLA-DR3/DR4 alleles predominated in T1AD patients. A heterozygous allelic IL-21 gene variant (g.-241 T > A) was found in only one patient. In conclusion, only PTPN22 C1858T polymorphism and HLA-DR3 and/or DR4 alleles, but not allelic variants in the 5'-proximal region of the IL-21 gene were associated with T1AD risk. Patients with T1AD had increased frequencies of anti-islet-cell, anti-thyroid, anti-nuclear, anti-smooth muscle and anti-21-OH autoantibodies. The C1858T PTPN22 polymorphism was also associated with a higher frequency of GAD65 and TG autoantibodies.

Relation of pretreatment sequence diversity in NS5A region of HCV genotype 1 with immune response between pegylated-INF/ribavirin therapy outcomes.

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific immune responses and the combination of pegylated interferon (INF)-α and ribavirin therapy. Major histocompatibility complex class I restricted CD8(+) T cells are responsible for the control of viraemia in HCV infection, and several studies suggest protection against viral infection associated with specific HLAs. The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Escape mutations in response to cytotoxic T lymphocyte are widely described; however, its influence in the treatment outcome is ill understood. Here, we investigate the differences in CD8 epitopes frequencies from the Los Alamos database between groups of patients that showed distinct response to pegylated α-INF with ribavirin therapy and test evidence of natural selection on the virus in those who failed treatment, using five maximum likelihood evolutionary models from PAML package. The group of sustained virological responders showed three epitopes with frequencies higher than Non-responders group, all had statistical support, and we observed evidence of selection pressure in the last group. No escape mutation was observed. Interestingly, the epitope VLSDFKTWL was 100% conserved in SVR group. These results suggest that the response to treatment can be explained by the increase in immune pressure, induced by interferon therapy, and the presence of those epitopes may represent an important factor in determining the outcome of therapy.

Relation of pretreatment sequence diversity in NS5A region ofHCV genotype 1 with immune response between pegylated- INF/ribavirin therapy outcomes

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific immune responsesand the combination of pegylated interferon (INF)-a and ribavirin therapy. Major histocompatibility complex class Irestricted CD8+ T cells are responsible for the control of viraemia in HCV infection, and several studies suggestprotection against viral infection associated with specific HLAs. The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Escape mutations in response to cytotoxic T lymphocyte are widely described; however, its influence in the treatment outcome is ill understood. Here, we investigate the differences in CD8 epitopes frequencies from the Los Alamos database between groups of patients that showed distinct response to pegylated a-INF with ribavirin therapy and test evidence of natural.

HTR1B and HTR2C in autism spectrum disorders in Brazilian families.

Autism spectrum disorders (ASD) is a group of behaviorally defined neurodevelopmental disabilities characterized by multiple genetic etiologies and a complex presentation. Several studies suggest the involvement of the serotonin system in the development of ASD, but only few have investigated serotonin receptors. We have performed a case-control and a family-based study with 9 polymorphisms mapped to two serotonin receptor genes (HTR1B and HTR2C) in 252 Brazilian male ASD patients of European ancestry. These analyses showed evidence of undertransmission of the HTR1B haplotypes containing alleles -161G and -261A at HTR1B gene to ASD (P=0.003), but no involvement of HTR2C to the predisposition to this disease. Considering the relatively low level of statistical significance and the power of our sample, further studies are required to confirm the association of these serotonin-related genes and ASD.

A framework for modeling of juxtacrine signaling systems.

Juxtacrine signaling is intercellular communication, in which the receptor of the signal (typically a protein) as well as the ligand (also typically a protein, responsible for the activation of the receptor) are anchored in the plasma membranes, so that in this type of signaling the activation of the receptor depends on direct contact between the membranes of the cells involved. Juxtacrine signaling is present in many important cellular events of several organisms, especially in the development process. We propose a generic formal model (a modeling framework) for juxtacrine signaling systems that is a class of discrete dynamic systems. It possesses desirable characteristics in a good modeling framework, such as: a) structural similarity with biological models, b) capacity of operating in different scales of time, and c) capacity of explicitly treating both the events and molecular elements that occur in the membrane, and those that occur in the intracellular environment and that are involved in the juxtacrine signaling process. We have implemented this framework and used it to develop a new three-level discrete model for the neurogenic network and its participation in neuroblast segregation. This paper presents the details of this framework and its current status.

Taxonomic and phylogenetic relationships between neotropical species of ticks from genus Amblyomma (Acari: Ixodidae) inferred from second internal transcribed spacer sequences of rDNA.

The accurate specific identification of ticks is essential for the study, control and prevention of tick-borne diseases. Herein, we determined ribosomal nucleotide sequences of the second internal transcribed spacer (ITS2) of 15 Neotropical hard tick species of the genus Amblyomma Koch found in Brazil. Most of the studied ticks accidentally parasite humans and potentially act as vectors of zoonoses. Lengths of the ITS2 sequences ranged from 956 to 1,207 bp, whereas GC content varied from 62.4 to 66.9%. A matrix of ITS2 divergence was calculated with the ITS2 sequence data obtained showing divergence levels varying from 1.5 to 28.8%. The analysis indicated that this molecular marker can be useful for Amblyomma-specific identification. Phylogenetic inferences based on the ITS2 sequences were used to assess some issues in subgenus taxonomy.

A framework for modeling of juxtacrine signaling systems

Juxtacrine signaling is intercellular communication, in which the receptor of the signal (typically a protein) as well as the ligand (also typically a protein, responsible for the activation of the receptor) are anchored in the plasma membranes, so that in this type of signaling the activation of the receptor depends on direct contact between the membranes of the cells involved. Juxtacrine signaling is present in many important cellular events of several organisms, especially in the development process. We propose a generic formal model (a modeling framework) for juxtacrine signaling systems that is a class of discrete dynamic systems. It possesses desirable characteristics in a good modeling framework, such as: a) structural similarity with biological models, b) capacity of operating in different scales of time, and c) capacity of explicitly treating both the events and molecular elements that occur in the membrane, and those that occur in the intracellular environment and that are involved in the juxtacrine signaling process. We have implemented this framework and used it to develop a new three-level discrete model for the neurogenic network and its participation in neuroblast segregation. This paper presents the details of this framework and its current status.

Epistasis affecting litter size in mice.

Litter size is an important reproductive trait as it makes a major contribution to fitness. Generally, traits closely related to fitness show low heritability perhaps because of the corrosive effects of directional natural selection on the additive genetic variance. Nonetheless, low heritability does not imply, necessarily, a complete absence of genetic variation because genetic interactions (epistasis and dominance) contribute to variation in traits displaying strong heterosis in crosses, such as litter size. In our study, we investigated the genetic architecture of litter size in 166 females from an F2 intercross of the SM/J and LG/J inbred mouse strains. Litter size had a low heritability (h2 = 12%) and a low repeatability (r = 33%). Using interval-mapping methods, we located two quantitative trait loci (QTL) affecting litter size at locations D7Mit21 + 0 cM and D12Mit6 + 8 cM, on chromosomes 7 and 12 respectively. These QTL accounted for 12.6% of the variance in litter size. In a two-way genome-wide epistasis scan we found eight QTL interacting epistatically involving chromosomes 2, 4, 5, 11, 14, 15 and 18. Taken together, the QTL and their interactions explain nearly 49% (39.5% adjusted multiple r2) of the phenotypic variation for litter size in this cross, an increase of 36% over the direct effects of the QTL. This indicates the importance of epistasis as a component of the genetic architecture of litter size and fitness in our intercross population.

Asymptomatic carriers and gender differences in facioscapulohumeral muscular dystrophy (FSHD).

Facioscapulohumeral muscular dystrophy is an autosomal dominant muscle disorder, mapped to 4q35. It is characterized by remarkable inter- and intrafamilial clinical variability ranging from severe phenotype to asymptomatic carriers. The aim of the present study was to assess the size of the Eco RI fragment in a large sample of asymptomatic or minimally affected carriers as well as symptomatic patients, comparing both sexes, in order to verify if asymptomatic carriers are randomly distributed or concentrated in some particular families and if there is preferential parental transmission (maternal or paternal) resulting in non-penetrant carriers. We have analysed a total of 506 individuals from 106 unrelated families with at least one affected facioscapulohumeral muscular dystrophy proband. In all patients the molecular diagnosis was confirmed following double digestion (Eco RI/Bln I fragment <35 kb). About 20% among probands' relatives who were found to carry the small fragment were asymptomatic or minimally affected, without preferential parental transmission, but with a significantly higher proportion of females (n=37) than males (n=14). Although asymptomatic carriers were found in about 30% of the families, some genealogies seem to concentrate more non-penetrant cases. A significant correlation between the size of the Eco RI fragment and severity of the phenotype was observed in the total sample but surprisingly this correlation is significant only among affected females. The gender difference in clinical manifestation as well as the observation that asymptomatic carriers are not rare should be taken into consideration in genetic counseling of affected patients or 'at-risk' relatives.

Evidence that BCL3 plays a role in the etiology of nonsyndromic oral clefts in Brazilian families.

The BCL3 gene has been considered a susceptibility locus for nonsyndromic cleft lip with or without cleft palate (NSCL/P), based on association and linkage studies in some populations. We evaluated an intragenic marker at the BCL3 gene and the microsatellite D19S178 (1.1 cM distant from the BCL3 gene) among 98 infants born with NSCL/P and their parents, using the transmission disequilibrium test (TDT) and a method for haplotype analysis. Our analysis, based on BCL3 alleles, revealed the existence of a marginal association of allele 135pb of the BCL3 gene with NSCL/P (chi(2)=3.60; P=0.058; 1 df), with a major effect in female (chi(2)=5.77; P=0.016; 1 df) and in familial cases (chi(2)=3.79; P=0.051; 1 df). However, the haplotype analysis detected no significant segregation distortion, even if the alleles of the D19S178 were grouped into two classes. These findings support previous findings that BCL3 plays a role in the etiology of NSCL/P as an allele of low penetrance or as a modifier locus. We hypothesize that there might be more than one mutation in this gene associated with NSCL/P, or alternatively, that more than one mutation has arisen associated with the 135-bp allele. Genet. Epidemiol. 23:364-374, 2002

No evidence of association between the D10S1423 locus and Alzheimer disease in Brazilian patients.

In a genome survey for Alzheimer's disease (AD), Zubenko et al. (1998) reported that the 234bp allele of the D10S1423 locus was more frequent among AD cases than in controls. We have analyzed this polymorphic locus in patients and healthy controls and observed that the 226bp allele is the most frequent allele in the D10S1423 locus in Brazilian AD patients. However, no statistically significant association between any D10S1423 allele was observed in AD patients as well as in controls.

Analysis of the disease risk locus DXS1047 polymorphism in Brazilian Alzheimer patients.

Alzheimer's disease (AD) is a disorder characterized by a progressive deterioration in memory and other cognitive functions. Four genes associated with early onset AD have been identified but familial AD is rare. The majority of late onset AD (LOAD) is caused by a complex inheritance with several genes interacting with environmental factors. The epsilon4 allele of the apolipoprotein E (APOE) gene has been reported worldwide as a risk factor associated with LOAD. The short variant of a polymorphism in the transcriptional region of the serotonin transporter gene (5-HTTLPR) was analyzed in several psychiatric conditions and found to be more frequently associated with European and Brazilian LOAD patients. Recently, allelic associations with LOAD were reported for five other loci, the most significant for one X-linked 202-bp allele, at the DXS1047 locus. We have analyzed this locus in Brazilian LOAD patients and observed that the 202-bp allele was not significantly more frequent among patients. In contrast, two other alleles (200 bp and 208 bp) were less frequent among AD male patients than in controls, confirming the importance of replicating association studies in different populations.

Coadapted gene complexes for morphological traits in Drosophila mercatorum. Two-loci interactions.

The availability of molecular and morphological markers in a parthenogenetically reproducing strain of Drosophila mercatorum allowed us to design an experiment in which we could obtain a sample of completely homozygous recombinant females from two different parental homozygous strains. The phenotypic values of body-size-related measures and bristle numbers (sternopleural and abdominal) were measured in females sampled from the parental, F1 and F2 generations. The DNA extracted from the F2 flies was scored for five Mendelian segregating loci through double stringency PCR. In addition, the flies were scored for three morphological recessive loci. We estimated all single-locus and all two-loci associations between the marker loci with the principal components of the morphological data, which allowed us also to estimate the epistatic parameters. The results suggest that the underlying genetic architecture of the morphological phenotypes cannot be regarded as a result of additivity only, but instead, involves many different kinds of interactions that are distributed around an additive mean.

Molecular adaptation of Drosophila melanogaster lysozymes to a digestive function.

A lysozyme (pI 5.5) was purified to homogeneity from heated acid extracts of Drosophila melanogaster larvae, using gel filtration in a Superose column and ion-exchange chromatography in a Mono Q column. The final yield was 67%. The purified lysozyme with Mr 13,700 (determined by SDS-polyacrylamide gel electrophoresis) decreases in activity and has its pH optimum displaced towards acidic values and Km increases as the ionic strength of the medium becomes higher. The lysozyme is resistant to a cathepsin D-like proteinase present in cyclorrhaphous Diptera and displays a chitinase activity which is 11-fold higher than that of chicken lysozyme. Microsequencing of an internal peptide of the purified lysozyme showed that this enzyme is the product of the previously sequenced Lys D gene. The results suggest that the product of the Lys P gene has pI 7.2, a pH optimum around 5 and is not a true digestive enzyme. The most remarkable sequence convergence of D. melanogaster lysozyme D and lysozymes from vertebrate foregut fermenters are serine 104 and a decrease in the number of basic amino acids, suggesting that these features are necessary for digestive function in an acid environment. Adaptive residues putatively conferring stability in an acid proteolytic environment differ between insects and vertebrates, probably because they depend on the overall three-dimensional structure of the lysozymes. A maximum likelihood phylogeny and inferences from insect lysozyme sequences showed that the recruitment of lysozymes as digestive enzymes is an ancestral condition of the flies (Diptera: Cyclorrhapha).

Obtaining genetic markers by using double-stringency PCR with microsatellites and arbitrary primers.

A technique to obtain genetic markers is described. PCR is performed at two different annealing temperatures in a single reaction mixture. During the first cycles, a single microsatellite oligonucleotide is used as primer to amplify DNA between two microsatellite regions. The annealing temperature and consequent stringency is then lowered, and in further cycles a random-amplified polymorphic DNA (RAPD) primer exponentially amplifies parts of the previously amplified fragments, which contain sequences complementary to the RAPD primer. The first round of specific amplification is found to be crucial for obtaining consistent, repeatable results, as compared with the results obtained by RAPD alone. This technique was proven to produce genetically informative markers, as revealed by crosses performed with Drosophila mercatorum strains, and it can be used for genetic mapping or population genetics studies.

Genetical and biochemical comparisons of alcohol dehydrogenase isozymes from Anastrepha fraterculus and A. obliqua (Diptera: Tephritidae): evidence for gene duplication.

The electrophoretic patterns of the enzyme alcohol dehydrogenase (ADH) from Anastrepha fraterculus and A. obliqua were studied. Two loci were found to code for the enzyme in A. fraterculus, and three in A. obliqua. In both species, all isozymes were active in third-instar larvae. A cationic isozyme (Adh-1) was active mainly in the visceral fat body of both species. In A. fraterculus, the locus had an anionic polymorphic isozyme (Adh-3) that was detected in the parietal fat body. In addition to these two loci, a third locus for an anionic isozyme (Adh-2), which was active in the digestive tube of larvae, was present in A. obliqua and probably resulted from gene duplication. For both species, multiple forms of the isozymes are formed by binding of an NAD-carbonyl compound, as in Drosophila melanogaster. Both larvae and early pupae of A. obliqua had almost twice the specific ADH activity as A. fraterculus. The ethanol content of the host fruit infested with A. obliqua (red "mombim") was also higher than that of the host fruit infested with A. fraterculus (guava).