Polymorphisms of the low-density lipoprotein receptor gene in Brazilian individuals with heterozygous familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a metabolic disorder inherited as an autosomal dominant trait characterized by an increased plasma low-density lipoprotein (LDL) level. The disease is caused by several different mutations in the LDL receptor gene. Although early identification of individuals carrying the defective gene could be useful in reducing the risk of atherosclerosis and myocardial infarction, the techniques available for determining the number of the functional LDL receptor molecules are difficult to carry out and expensive. Polymorphisms associated with this gene may be used for unequivocal diagnosis of FH in several populations. The aim of our study was to evaluate the genotype distribution and relative allele frequencies of three polymorphisms of the LDL receptor gene, HincII1773 (exon 12), AvaII (exon 13) and PvuII (intron 15), in 50 unrelated Brazilian individuals with a diagnosis of heterozygous FH and in 130 normolipidemic controls. Genomic DNA was extracted from blood leukocytes by a modified salting-out method. The polymorphisms were detected by PCR-RFLP. The FH subjects showed a higher frequency of A+A+ (AvaII), H+H+ (HincII1773) and P1P1 (PvuII) homozygous genotypes when compared to the control group (P<0.05). In addition, FH probands presented a high frequency of A+ (0.58), H+ (0.61) and P1 (0.78) alleles when compared to normolipidemic individuals (0.45, 0.45 and 0.64, respectively). The strong association observed between these alleles and FH suggests that AvaII, HincII1773 and PvuII polymorphisms could be useful to monitor the inheritance of FH in Brazilian families.

The eukaryotic Pso2/Snm1/Artemis proteins and their function as genomic and cellular caretakers

DNA double-strand breaks (DSBs) represent a major threat to the genomic stability of eukaryotic cells. DNA repair mechanisms such as non-homologous end joining (NHEJ) are responsible for the maintenance of eukaryotic genomes. Dysfunction of one or more of the many protein complexes that function in NHEJ can lead to sensitivity to DNA damaging agents, apoptosis, genomic instability, and severe combined immunodeficiency. One protein, Pso2p, was shown to participate in the repair of DSBs induced by DNA inter-strand cross-linking (ICL) agents such as cisplatin, nitrogen mustard or photo-activated bi-functional psoralens. The molecular function of Pso2p in DNA repair is unknown, but yeast and mammalian cell line mutants for PSO2 show the same cellular responses as strains with defects in NHEJ, e.g., sensitivity to ICLs and apoptosis. The Pso2p human homologue Artemis participates in V(D)J recombination. Mutations in Artemis induce a variety of immunological deficiencies, a predisposition to lymphomas, and an increase in chromosomal aberrations. In order to better understand the role of Pso2p in the repair of DSBs generated as repair intermediates of ICLs, an in silico approach was used to characterize the catalytic domain of Pso2p, which led to identification of novel Pso2p homologues in other organisms. Moreover, we found the catalytic core of Pso2p fused to different domains. In plants, a specific ATP-dependent DNA ligase I contains the catalytic core of Pso2p, constituting a new DNA ligase family, which was named LIG6. The possible functions of Pso2p/Artemis/Lig6p in NHEJ and V(D)J recombination and in other cellular metabolic reactions are discussed.

Genomic analysis of Brazilian patients with Fabry disease

Fabry disease is an X-linked lysosomal disorder due to a-galactosidase A deficiency that causes storage of globotriaosylceramide. The gene coding for this lysosomal enzyme is located on the long arm of the X chromosome, in region Xq21.33-Xq22. Disease progression leads to vascular disease secondary to involvement of kidney, heart and the central nervous system. Detection of female carriers based solely on enzyme assays is often inconclusive. Therefore, mutation analysis is a valuable tool for diagnosis and genetic counseling. Many mutations of the a-galactosidase A gene have been reported with high genetic heterogeneity, being most mutations private found in only one family. The disease is panethnic, and estimates of incidence range from about 1 in 40,000 to 60,000 males. Our objective was to describe the analysis of 6 male and 7 female individuals belonging to 4 different Fabry disease families by automated sequencing of the seven exons of the a-galactosidase gene. Sequencing was performed using PCR fragments for each exon amplified from DNA extracted from peripheral blood. Three known mutations and one previously described in another Brazilian family were detected. Of 7 female relatives studied, 4 were carriers. Although the present study confirms the heterogeneity of mutations in Fabry disease, the finding of the same mutation previously detected in another Fabry family from our region raises the possibility of some founder effect, or genetic drift. Finally, the present study highlights the importance of molecular analysis for carrier detection and genetic counseling.

DNA tests probe the genomic ancestry of Brazilians

We review studies from our laboratories using different molecular tools to characterize the ancestry of Brazilians in reference to their Amerindian, European and African roots. Initially we used uniparental DNA markers to investigate the contribution of distinct Y chromosome and mitochondrial DNA lineages to present-day populations. High levels of genetic admixture and strong directional mating between European males and Amerindian and African females were unraveled. We next analyzed different types of biparental autosomal polymorphisms. Especially useful was a set of 40 insertion-deletion polymorphisms (indels) that when studied worldwide proved exquisitely sensitive in discriminating between Amerindians, Europeans and Sub-Saharan Africans. When applied to the study of Brazilians these markers confirmed extensive genomic admixture, but also demonstrated a strong imprint of the massive European immigration wave in the 19th and 20th centuries. The high individual ancestral variability observed suggests that each Brazilian has a singular proportion of Amerindian, European and African ancestries in his mosaic genome. In Brazil, one cannot predict the color of persons from their genomic ancestry nor the opposite. Brazilians should be assessed on a personal basis, as 190 million human beings, and not as members of color groups.

Genomic alterations detected by comparative genomic hybridization in ovarian endometriomas

Endometriosis is a complex and multifactorial disease. Chromosomal imbalance screening in endometriotic tissue can be used to detect hot-spot regions in the search for a possible genetic marker for endometriosis. The objective of the present study was to detect chromosomal imbalances by comparative genomic hybridization (CGH) in ectopic tissue samples from ovarian endometriomas and eutopic tissue from the same patients. We evaluated 10 ovarian endometriotic tissues and 10 eutopic endometrial tissues by metaphase CGH. CGH was prepared with normal and test DNA enzymatically digested, ligated to adaptors and amplified by PCR. A second PCR was performed for DNA labeling. Equal amounts of both normal and test-labeled DNA were hybridized in human normal metaphases. The Isis FISH Imaging System V 5.0 software was used for chromosome analysis. In both eutopic and ectopic groups, 4/10 samples presented chromosomal alterations, mainly chromosomal gains. CGH identified 11q12.3-q13.1, 17p11.1-p12, 17q25.3-qter, and 19p as critical regions. Genomic imbalances in 11q, 17p, 17q, and 19p were detected in normal eutopic and/or ectopic endometrium from women with ovarian endometriosis. These regions contain genes such as POLR2G, MXRA7 and UBA52 involved in biological processes that may lead to the establishment and maintenance of endometriotic implants. This genomic imbalance may affect genes in which dysregulation impacts both eutopic and ectopic endometrium.

Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated.

Mutation and genomic amplification of the PIK3CA proto-oncogene in pituitary adenomas

The tumorigenesis of pituitary adenomas is poorly understood. Mutations of the PIK3CA proto-oncogene, which encodes the p110-α catalytic subunit of PI3K, have been reported in various types of human cancers regarding the role of the gene in cell proliferation and survival through activation of the PI3K/Akt signaling pathway. Only one Chinese study described somatic mutations and amplification of the PIK3CA gene in a large series of pituitary adenomas. The aim of the present study was to determine genetic alterations of PIK3CA in a second series that consisted of 33 pituitary adenomas of different subtypes diagnosed by immunohistochemistry: 6 adrenocorticotropic hormone-secreting microadenomas, 5 growth hormone-secreting macroadenomas, 7 prolactin-secreting macroadenomas, and 15 nonfunctioning macroadenomas. Direct sequencing of exons 9 and 20 assessed by qPCR was employed to investigate the presence of mutations and genomic amplification defined as a copy number ≥4. Previously identified PIK3CA mutations (exon 20) were detected in four cases (12.1%). Interestingly, the Chinese study reported mutations only in invasive tumors, while we found a PIK3CA mutation in one noninvasive corticotroph microadenoma. PIK3CA amplification was observed in 21.2% (7/33) of the cases. This study demonstrates the presence of somatic mutations and amplifications of the PIK3CA gene in a second series of pituitary adenomas, corroborating the previously described involvement of the PI3K/Akt signaling pathway in the tumorigenic process of this gland.

Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting thatXIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance ofXIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.

Caryocar brasiliense camb protects against genomic and oxidative damage in urethane-induced lung carcinogenesis

The antioxidant effects of Caryocar brasiliense Camb, commonly known as the pequi fruit, have not been evaluated to determine their protective effects against oxidative damage in lung carcinogenesis. In the present study, we evaluated the role of pequi fruit against urethane-induced DNA damage and oxidative stress in forty 8-12 week old male BALB/C mice. An in vivo comet assay was performed to assess DNA damage in lung tissues and changes in lipid peroxidation and redox cycle antioxidants were monitored for oxidative stress. Prior supplementation with pequi oil or its extract (15 µL, 60 days) significantly reduced urethane-induced oxidative stress. A protective effect against DNA damage was associated with the modulation of lipid peroxidation and low protein and gene expression of nitric oxide synthase. These findings suggest that the intake of pequi fruit might protect against in vivo genotoxicity and oxidative stress.

Effects of Obesity and Weight Loss Following Bariatric Surgery on Brain Function, Structural Integrity and Metabolism

Obesity is one of the key challenges to health care system worldwide and its prevalence is estimated to rise to pandemic proportions. Numerous adverse health effects follow with increasing body weight, including increased risk of hypertension, diabetes, hypercholesterolemia, musculoskeletal pain and cancer. Current evidence suggests that obesity is associated with altered cerebral reward circuit functioning and decreased inhibitory control over appetitive food cues. Furthermore, obesity causes adverse shifts in metabolism and loss of structural integrity within the brain. Prior cross-sectional studies do not allow delineating which of these cerebral changes are recoverable after weight loss. We compared morbidly obese subjects with healthy controls to unravel brain changes associated with obesity. Bariatric surgery was used as an intervention to study which cerebral changes are recoverable after weight loss. In Study I we employed functional magnetic resonance imaging (fMRI) to detect the brain basis of volitional appetite control and its alterations in obesity. In Studies II-III we used diffusion tensor imaging (DTI) and voxel-based morphometry (VBM) to quantify the effects of obesity and the effects of weight loss on structural integrity of the brain. In study IV we used positron emission tomography (PET) with [18F]-FDG in fasting state and during euglycemic hyperinsulinemia to quantify effects of obesity and weight loss on brain glucose uptake. The fMRI experiment revealed that a fronto-parietal network is involved in volitional appetite control. Obese subjects had lower medial frontal and dorsal striatal brain activity during cognitive appetite control and increased functional connectivity within the appetite control circuit. Obese subjects had initially lower grey matter and white matter densities than healthy controls in VBM analysis and loss of integrity in white matter tracts as measured by DTI. They also had initially elevated glucose metabolism under insulin stimulation but not in fasting state. After the weight loss following bariatric surgery, obese individuals’ brain volumes recovered and the insulin-induced increase in glucose metabolism was attenuated. In conclusion, obesity is associated with altered brain function, coupled with loss of structural integrity and elevated glucose metabolism, which are likely signs of adverse health effects to the brain. These changes are reversed by weight loss after bariatric surgery, implicating that weight loss has a causal role on these adverse cerebral changes. Altogether these findings suggest that weight loss also promotes brain health.Key words: brain, obesity, bariatric surgery, appetite control, structural magnetic resonance

Genetics of inherited small vessel diseases – in search of a novel small vessel disease and modifiers of the clinical course of CADASIL

The genetic and environmental risk factors of vascular cognitive impairment are still largely unknown. This thesis aimed to assess the genetic background of two clinically similar familial small vessel diseases (SVD), CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) and Swedish hMID (hereditary multi-infarct dementia of Swedish type). In the first study, selected genetic modifiers of CADASIL were studied in a homogenous Finnish CADASIL population of 134 patients, all carrying the p.Arg133Cys mutation in NOTCH3. Apolipoprotein E (APOE) genotypes, angiotensinogen (AGT) p.Met268Thr polymorphism and eight NOTCH3 polymorphisms were studied, but no associations between any particular genetic variant and first-ever stroke or migraine were seen. In the second study, smoking, statin medication and physical activity were suggested to be the most profound environmental differences among the monozygotic twins with CADASIL. Swedish hMID was for long misdiagnosed as CADASIL. In the third study, the CADASIL diagnosis in the Swedish hMID family was ruled out on the basis of genetic, radiological and pathological findings, and Swedish hMID was suggested to represent a novel SVD. In the fourth study, the gene defect of Swedish hMID was then sought using whole exome sequencing paired with a linkage analysis. The strongest candidate for the pathogenic mutation was a 3’UTR variant in the COL4A1 gene, but further studies are needed to confirm its functionality. This study provided new information about the genetic background of two inherited SVDs. Profound knowledge about the pathogenic mutations causing familial SVD is also important for correct diagnosis and treatment options.

Characterisation of Cladonia rangiferina transcriptome and genome, and the effects of dehydration and rehydration on its gene expression

Lichens are symbiotic organisms, which consist of the fungal partner and the photosynthetic partner, which can be either an alga or a cyanobacterium. In some lichen species the symbiosis is tripartite, where the relationship includes both an alga and a cyanobacterium alongside the primary symbiont, fungus. The lichen symbiosis is an evolutionarily old adaptation to life on land and many extant fungal species have evolved from lichenised ancestors. Lichens inhabit a wide range of habitats and are capable of living in harsh environments and on nutrient poor substrates, such as bare rocks, often enduring frequent cycles of drying and wetting. Most lichen species are desiccation tolerant, and they can survive long periods of dehydration, but can rapidly resume photosynthesis upon rehydration. The molecular mechanisms behind lichen desiccation tolerance are still largely uncharacterised and little information is available for any lichen species at the genomic or transcriptomic level. The emergence of the high-throughput next generation sequencing (NGS) technologies and the subsequent decrease in the cost of sequencing new genomes and transcriptomes has enabled non-model organism research on the whole genome level. In this doctoral work the transcriptome and genome of the grey reindeer lichen, Cladonia rangiferina, were sequenced, de novo assembled and characterised using NGS and traditional expressed sequence tag (EST) technologies. RNA extraction methods were optimised to improve the yield and quality of RNA extracted from lichen tissue. The effects of rehydration and desiccation on C. rangiferina gene expression on whole transcriptome level were studied and the most differentially expressed genes were identified. The secondary metabolites present in C. rangiferina decreased the quality – integrity, optical characteristics and utility for sensitive molecular biological applications – of the extracted RNA requiring an optimised RNA extraction method for isolating sufficient quantities of high-quality RNA from lichen tissue in a time- and cost-efficient manner. The de novo assembly of the transcriptome of C. rangiferina was used to produce a set of contiguous unigene sequences that were used to investigate the biological functions and pathways active in a hydrated lichen thallus. The de novo assembly of the genome yielded an assembly containing mostly genes derived from the fungal partner. The assembly was of sufficient quality, in size similar to other lichen-forming fungal genomes and included most of the core eukaryotic genes. Differences in gene expression were detected in all studied stages of desiccation and rehydration, but the largest changes occurred during the early stages of rehydration. The most differentially expressed genes did not have any annotations, making them potentially lichen-specific genes, but several genes known to participate in environmental stress tolerance in other organisms were also identified as differentially expressed.

Desiccation tolerance and DNA integrity in Eugenia pleurantha O. Berg. (myrtaceae) seeds

The aim of this study was to assess the desiccation tolerance and DNA integrity in Eugenia pleurantha seeds dehydrated to different moisture contents (MCs). Seeds extracted from mature fruits were submmited to drying in silica gel and evaluated at every five percentual points of decrease from the initial MC (35.5%, fresh weight basis). The effects of dehydration on seeds were verified through germination tests and DNA integrity assessment. Undried seeds achieved 87% germination, value reduced to 36% after being dried to 9.8% MC. When dried slightly more, to 7.4% MC, seeds were no longer able to germinate, suggesting an intermediate behavior in relation to desiccation tolerance. It was observed DNA degradation in seeds with 7.4% MC, which might have contributed to the loss of seed germination.

Desiccation tolerance and dna integrity in Eugenia pleurantha o. berg. (Myrtaceae) seeds

The aim of this study was to assess the desiccation tolerance and DNA integrity in Eugenia pleurantha seeds dehydrated to different moisture contents (MCs). Seeds extracted from mature fruits were dried in silica gel and evaluated at every five percentual points of decrease from the initial MC (35.5%, fresh weight basis). The effects of dehydration on seeds were verified through germination tests and DNA integrity assessment. Undried seeds achieved 87% germination, value reduced to 36% after being dried to 9.8% MC. When dried slightly more, to 7.4% MC, seeds were no longer able to germinate, suggesting an intermediate behavior in relation to desiccation tolerance. DNA degradation was observed in seeds with 7.4% MC, which might have contributed to the loss of seed germination.

Cloning of actin genes from a genomic library of the newt, Notophthalmus viridescens

The regenerating urodele limb is a useful model system in which to study, in vivo, the controls of cell proliferation and differentiation. Techniques are available which enable one to experimentally manipulate mitogenic influences upon the blastema, as well the morphogenesis of the regenerating 11mb. Although classical regeneration studies have generated a wealth of knowledge concerning tissue interactions, little 1s known about the process at the level of gene expression. The aim of this project was to clone potentially developmentally regulated genes from a newt genomic library for use in future studies of gene expression during limb regeneration. We decided to clone the cytoskeletal actin gene for the following reasons: 1. its expression reflects the proliferative and differentiatlve states of cells in other systems 2. the high copy number of cytoplasmic actin pseudogenes in other vertebrates and the high degree of evolutionary sequence conservation among actin genes increased the chance of cloning one of the newt cytoplasmic actin genes. 3. Preliminary experiments indicated that a newt actin could probably be identified using an available chick ~-actln gene for a molecular probe. Two independent recombinant phage clones, containing actin homologous inserts, were isolated from a newt genomic library by hybridization with the chick actin probe. Restriction mapping identified actin homologous sequences within the newt DNA inserts which were subcloned into the plasmid pTZ19R. The recombinant plasmids were transformed into the Escherichia coli strain, DHsa. Detailed restriction maps were produced of the 5.7Kb and 3.1Kb newt DNA inserts in the plasmids, designated pTNAl and pTNA2. The short «1.3 Kb) length of the actin homologous sequence in pTNA2 indicated that it was possibly a reverse transcript pseudogene. Problems associated with molecular cloning of DNA sequences from N. viridescens are discussed with respect to the large genome size and abundant highly repetitive DNA sequences.