Genetic predisposition to prostate cancer

Prostate cancer (PC) is a significant economic and health burden in the U.S. and Europe but its causes are largely unknown. The most significant risk factors (after gender) are age and family history of the disease. A gene with high penetrance but low frequency on chromosome 1q, HPC 1, has been suggested to cause a proportion of the familial aggregation of PC but other more common genes, conferring less risk, are also thought to contribute to disease predisposition. We have pursued a strategy to study both types of genetic risk in PC. To identify high penetrance genes, affected men from thirteen families have been genotyped for genetic linkage analysis at six microsatellite markers spanning 45 cM of 1q24-25. Both LOD score and non-parametric statistics provide no significant support for HPC1 in this genomic region, although 3 of the families did combine to produce a LOD score of 0.9. These families will be included in a genome wide search for other PC predisposition genes as part of a multinational collaboration.^ For study of common genetic factors in PC development, leukocyte DNA samples from an unselected series of 55 patients and 67 controls have been examined for genetic differences in two other candidate genes, the androgen receptor gene, hAR, at Xq11-12, and the vitamin D receptor gene, hVDR, at 12q12-14. hAR was typed for two trinucleotide repeat length polymorphisms, (CAG)$\rm\sb{n}$ and (GGC)$\rm\sb{n},$ encoding polyglutamine and polyglycine tracts, respectively, which have been implicated in PC susceptibility. These data, combined with similarly processed patients and controls from the U.K. show no consistent association of allele length with PC risk. A novel finding, however, has been a significant association between the number of GGC repeats and the length of time between diagnosis and relapse in stage T1-T4 Caucasian patients irrespective of therapy and age of the patient. Of 49 patients who relapsed out of 108 entering the study, those with 16 or fewer GGC repeats had an average relapse-free-period of 101 (+/$-$7.7) months while for those with more than 16 repeats the period averaged 48 (+/$-$2.9) months, a difference of 2.1 fold or 4.4 years.^ The second gene, hVDR, was genotyped at two polymorphisms, a synonymous C/T substitution in exon 9 identified by differential TaqI enzymatic digestion and a variable length polyA tract in the 3$\sp\prime$ UTR. Although these polymorphisms are in strong linkage disequilibrium only the polyA region showed a possible association with PC risk. Men homozygous for alleles with fewer than 18 A's had an increased risk (OR = 3.0, p = 0.0578) compared to controls. This result is opposite to the findings of others and may either indicate off-setting random errors which together balance out to no significant overall effect or reflect more complex genetic and/or environmental associations.^ Overall, this research suggests that single gene familial predisposition may be less prominent in PC than in other cancers and that the characteristics of PC pathology may be useful in identifying the effects of common genetic factors. ^

4-year clinical outcomes and predictors of repeat revascularization in patients treated with new-generation drug-eluting stents: a report from the RESOLUTE All-Comers trial (A Randomized Comparison of a Zotarolimus-Eluting Stent With an Everolimus-Eluting Stent for Percutaneous Coronary Intervention)

OBJECTIVES The aim of the study was to investigate 4-year outcomes and predictors of repeat revascularization in patients treated with the Resolute zotarolimus-eluting stent (R-ZES) (Medtronic, Minneapolis, Minnesota) and XIENCE V everolimus-eluting stent (EES) (Abbott Vascular, Abbott Park, Illinois) in the RESOLUTE (A Randomized Comparison of a Zotarolimus-Eluting Stent With an Everolimus-Eluting Stent for Percutaneous Coronary Intervention) All-Comers trial. BACKGROUND Data on long-term outcomes of new-generation drug-eluting stents are limited, and predictors of repeat revascularization due to restenosis and/or progression of disease are largely unknown. METHODS Patients were randomly assigned to treatment with the R-ZES (n = 1,140) or the EES (n = 1,152). We assessed pre-specified safety and efficacy outcomes at 4 years including target lesion failure and stent thrombosis. Predictors of revascularization at 4 years were identified by Cox regression analysis. RESULTS At 4 years, the rates of target lesion failure (15.2% vs. 14.6%, p = 0.68), cardiac death (5.4% vs. 4.7%, p = 0.44), and target vessel myocardial infarction (5.3% vs. 5.4%, p = 1.00), clinically-indicated target lesion revascularization (TLR) (7.0% vs. 6.5%, p = 0.62), and definite/probable stent thrombosis (2.3% vs. 1.6%, p = 0.23) were similar with the R-ZES and EES. Independent predictors of TLR were age, insulin-treated diabetes, SYNTAX (Synergy between PCI with Taxus and Cardiac Surgery) score, treatment of saphenous vein grafts, ostial lesions, and in-stent restenosis. Independent predictors of any revascularization were age, diabetes, previous percutaneous coronary intervention, absence of ST-segment elevation myocardial infarction, smaller reference vessel diameter, SYNTAX score, and treatment of left anterior descending, right coronary artery, saphenous vein grafts, ostial lesions, or in-stent restenosis. CONCLUSIONS R-ZES and EES demonstrated similar safety and efficacy throughout 4 years. TLR represented less than one-half of all repeat revascularization procedures. Patient- and lesion-related factors predicting the risk of TLR and any revascularization showed considerable overlap. (A Randomized Comparison of a Zotarolimus-Eluting Stent With an Everolimus-Eluting Stent for Percutaneous Coronary Intervention [RESOLUTE-AC]; NCT00617084).

Treatment of exudative age-related macular degeneration with a designed ankyrin repeat protein that binds vascular endothelial growth factor: a phase I/II study

PURPOSE To evaluate the safety, tolerability and bioactivity of ascending doses of MP0112, a designed ankyrin repeat protein (DARPin) that binds with high affinity to vascular endothelial growth factor-A (VEGF-A), in treatment-naive patients with exudative age-related macular degeneration (AMD). DESIGN Phase I/II, open-label, multicenter, dose-escalation study. METHODS Patients were to receive a single intravitreal injection of MP0112 at doses ranging from 0.04 to 3.6 mg and be monitored for 16 weeks for safety, efficacy, pharmacokinetics, and dose response. RESULTS Altogether, 32 patients received a single injection of MP0112. The maximum tolerated dose was 1.0 mg because of a case of endophthalmitis in the 2.0 mg cohort. Drug-related adverse events were reported by 13 (41%) of 32 patients; they included ocular inflammation in 11 patients (7 mild, 4 moderate in severity). Visual acuity scores were stable or improved compared with baseline for ≥4 weeks following injection; both retinal thickness and fluorescein angiography leakage decreased in a dose-dependent manner. Rescue therapy was administered to 20 (91%) of 22 patients who received 0.04-0.4 mg MP0112 compared with 4 of 10 (40%) patients who received 1.0 or 2.0 mg. Of patients in the higher-dose cohorts who did not require rescue treatment, 83% (5/6) maintained reductions in central retinal thickness through week 16. CONCLUSIONS A single injection of 1.0 or 2.0 mg MP0112 resulted in mean decreases in retinal thickness and leakage area despite ocular inflammation. Larger-scale studies are warranted to confirm these observations.

Genetic epidemiology of gallbladder disease in Mexican Americans and cholesterol 7$\alpha$-hydroxylase gene sequence variation

Among Mexican Americans, the second largest minority group in the United States, the prevalence of gallbladder disease is markedly elevated. Previous data from both genetic admixture and family studies indicate that there is a genetic component to the occurrence of gallbladder disease in Mexican Americans. However, prior to this thesis no formal genetic analysis of gallbladder disease had been carried out nor had any contributing genes been identified.^ The results of complex segregation analysis in a sample of 232 Mexican American pedigrees documented the existence of a major gene having two alleles with age- and gender-specific effects influencing the occurrence of gallbladder disease. The estimated frequency of the allele increasing susceptibility was 0.39. The lifetime probabilities that an individual will be affected by gallbladder disease were 1.0, 0.54, and 0.00 for females of genotypes "AA", "Aa", and "aa", respectively, and 0.68, 0.30, and 0.00 for males, respectively. This analysis provided the first conclusive evidence for the existence of a common single gene having a large effect on the occurrence of gallbladder disease.^ Human cholesterol 7$\alpha$-hydroxylase is the rate-limiting enzyme in bile acid synthesis. The results of an association study in both a random sample and a matched case/control sample showed that there is a significant association between cholesterol 7$\alpha$-hydroxylase gene variation and the occurrence of gallbladder disease in Mexican Americans males but not in females. These data have implicated a specific gene, 7$\alpha$-hydroxylase, in the etiology of gallbladder disease in this population.^ Finally, I asked whether the inferred major gene from complex segregation analysis is genetically linked to the cholesterol 7$\alpha$-hydroxylase gene. Three pedigrees predicted to be informative for linkage analysis by virtue of supporting the major gene hypothesis and having parents with informative genotypes and multiple offspring were selected for this linkage analysis. In each of these pedigrees, the recombination fractions maximized at 0 with a positive, albeit low, LOD score. The results of this linkage analysis provide preliminary and suggestive evidence that the cholesterol 7$\alpha$-hydroxylase gene and the inferred gallbladder disease susceptibility gene are genetically linked. ^

Gilbert syndrome and glucose-6-phosphate dehydrogenase deficiency: A dose-dependent genetic interaction crucial to neonatal hyperbilirubinemia

Severe jaundice leading to kernicterus or death in the newborn is the most devastating consequence of glucose-6-phosphate dehydrogenase (EC 1.1.1.49; G-6-PD) deficiency. We asked whether the TA repeat promoter polymorphism in the gene for uridinediphosphoglucuronate glucuronosyltransferase 1 (EC 2.4.1.17; UDPGT1), associated with benign jaundice in adults (Gilbert syndrome), increases the incidence of neonatal hyperbilirubinemia in G-6-PD deficiency. DNA from term neonates was analyzed for UDPGT1 polymorphism (normal homozygotes, heterozygotes, variant homozygotes), and for G-6-PD Mediterranean deficiency. The variant UDPGT1 promoter allele frequency was similar in G-6-PD-deficient and normal neonates. Thirty (22.9%) G-6-PD deficient neonates developed serum total bilirubin ≥ 257 μmol/liter, vs. 22 (9.2%) normals (P = 0.0005). Of those with the normal homozygous UDPGT1 genotype, the incidence of hyperbilirubinemia was similar in G-6-PD-deficients and controls (9.7% and 9.9%). In contrast, in the G-6-PD-deficient neonates, those with the heterozygous or homozygous variant UDPGT1 genotype had a higher incidence of hyperbilirubinemia than corresponding controls (heterozygotes: 31.6% vs. 6.7%, P < 0.0001; variant homozygotes: 50% vs. 14.7%, P = 0.02). Among G-6-PD-deficient infants the incidence of hyperbilirubinemia was greater in those with the heterozygous (31.6%, P = 0.006) or variant homozygous (50%, P = 0.003) UDPGT1 genotype than in normal homozygotes. In contrast, among those normal for G-6-PD, the UDPGT1 polymorphism had no significant effect (heterozygotes: 6.7%; variant homozygotes: 14.7%). Thus, neither G-6-PD deficiency nor the variant UDPGT1 promoter, alone, increased the incidence of hyperbilirubinemia, but both in combination did. This gene interaction may serve as a paradigm of the interaction of benign genetic polymorphisms in the causation of disease.

Three novel families of miniature inverted-repeat transposable elements are associated with genes of the yellow fever mosquito, Aedes aegypti

Three novel families of transposable elements, Wukong, Wujin, and Wuneng, are described in the yellow fever mosquito, Aedes aegypti. Their copy numbers range from 2,100 to 3,000 per haploid genome. There are high degrees of sequence similarity within each family, and many structural but not sequence similarities between families. The common structural characteristics include small size, no coding potential, terminal inverted repeats, potential to form a stable secondary structure, A+T richness, and putative 2- to 4-bp A+T-biased specific target sites. Evidence of previous mobility is presented for the Wukong elements. Elements of these three families are associated with 7 of 16 fully or partially sequenced Ae. aegypti genes. Characteristics of these mosquito elements indicate strong similarities to the miniature inverted-repeat transposable elements (MITEs) recently found to be associated with plant genes. MITE-like elements have also been reported in two species of Xenopus and in Homo sapiens. This characterization of multiple families of highly repetitive MITE-like elements in an invertebrate extends the range of these elements in eukaryotic genomes. A hypothesis is presented relating genome size and organization to the presence of highly reiterated MITE families. The association of MITE-like elements with Ae. aegypti genes shows the same bias toward noncoding regions as in plants. This association has potentially important implications for the evolution of gene regulation.

Equilibrium distributions of microsatellite repeat length resulting from a balance between slippage events and point mutations

We describe and test a Markov chain model of microsatellite evolution that can explain the different distributions of microsatellite lengths across different organisms and repeat motifs. Two key features of this model are the dependence of mutation rates on microsatellite length and a mutation process that includes both strand slippage and point mutation events. We compute the stationary distribution of allele lengths under this model and use it to fit DNA data for di-, tri-, and tetranucleotide repeats in humans, mice, fruit flies, and yeast. The best fit results lead to slippage rate estimates that are highest in mice, followed by humans, then yeast, and then fruit flies. Within each organism, the estimates are highest in di-, then tri-, and then tetranucleotide repeats. Our estimates are consistent with experimentally determined mutation rates from other studies. The results suggest that the different length distributions among organisms and repeat motifs can be explained by a simple difference in slippage rates and that selective constraints on length need not be imposed.

SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis.

Gibberellins (GAs) are a major class of plant hormones that control many developmental processes, including seed development and germination, flower and fruit development, and flowering time. Genetic studies with Arabidopsis thaliana have identified two genes involved in GA perception or signal transduction. A semidominant mutation at the GIBBERELLIN INSENSITIVE (GAI) locus results in plants resembling GA-deficient mutants but exhibiting reduced sensitivity to GA. Recessive mutations at the SPINDLY (SPY) locus cause a phenotype that is consistent with constitutive activation of GA signal transduction. Here we show that a strong allele of spy is completely epistatic to gai, indicating that SPY acts downstream of GAI. We have cloned the SPY gene and shown that it encodes a new type of signal transduction protein, which contains a tetratricopeptide repeat region, likely serving as a protein interaction domain, and a novel C-terminal region. Mutations in both domains increase GA signal transduction. The presence of a similar gene in Caenorhabditis elegans suggests that SPY represents a class of signal transduction proteins that is present throughout the eukaryotes.

Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development.

The Ah receptor (AHR) is a ligand-activated transcription factor that mediates a pleiotropic response to environmental contaminants such as benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. In an effort to gain insight into the physiological role of the AHR and to develop models useful in risk assessment, gene targeting was used to inactivate the murine Ahr gene by homologous recombination. Ahr-/- mice are viable and fertile but show a spectrum of hepatic defects that indicate a role for the AHR in normal liver growth and development. The Ahr-/- phenotype is most severe between 0-3 weeks of age and involves slowed early growth and hepatic defects, including reduced liver weight, transient microvesicular fatty metamorphosis, prolonged extramedullary hematopoiesis, and portal hypercellularity with thickening and fibrosis.

Expansion of polyglutamine repeat in huntingtin leads to abnormal protein interactions involving calmodulin.

Huntington's disease (HD) is an inherited neurodegenerative disorder associated with expansion of a CAG repeat in the IT15 gene. The IT15 gene is translated to a protein product termed huntingtin that contains a polyglutamine (polyGln) tract. Recent investigations indicate that the cause of HD is expansion of the polyGln tract. However, the function of huntingtin and how the expanded polyGln tract causes HD is not known. We investigate potential protein-protein interactions of huntingtin using affinity resins. Huntingtin from brain extracts is retained on calmodulin(CAM)-Sepharose in a calcium-dependent fashion. We purify rat huntingtin to apparent homogeneity using a combination of DEAE-cellulose column chromatography, ammonium sulfate precipitation, and preparative SDS/PAGE. Purified rat huntingtin does not interact with CAM directly as revealed by 125I-CAM overlay. Huntingtin forms a large CAM-containing complex of over 1,000 kDa in the presence of calcium, which partially disassociates in the absence of calcium. Furthermore, an increased amount of mutant huntingtin from HD patient brains is retained on CAM-Sepharose compared to normal huntingtin from control patient brains, and the mutant allele is preferentially retained on CAM-Sepharose in the absence of calcium. These results suggest that huntingtin interacts with other proteins including CAM and that the expansion of polyGln alters this interaction.

An increased risk of Crohn's disease in individuals who inherit the HLA class II DRB3*0301 allele.

The role of inflammatory T cells in Crohn's disease suggests that inherited variations in major histocompatibility complex (MHC) class II genes may be of pathogenetic importance in inflammatory bowel disease. The absence of consistent and strong associations with MHC class II genes in Caucasian patients with inflammatory bowel disease probably reflects the use of less precise typing approaches and the failure to type certain loci by any means. A PCR-sequence-specific oligonucleotide-based approach was used to type individual alleles of the HLA class II DRB1, DRB3, DRB4, and DRB5 loci in 40 patients with ulcerative colitis, 42 Crohn's disease patients, and 93 ethnically matched healthy controls. Detailed molecular typing of the above alleles has previously not been reported in patients with inflammatory bowel disease. A highly significant positive association with the HLA-DRB3*0301 allele was observed in patients with Crohn's disease (P = 0.0004) but not in patients with ulcerative colitis. The relative risk for this association was 7.04. Other less significant HLA class II associations were also noted in patients with Crohn's disease. One of these associations involved the HLA-DRB1*1302 allele, which is known to be in linkage disequilibrium with HLA-DRB3*0301. These data suggest that a single allele of an infrequently typed HLA class II locus is strongly associated with Crohn's disease and that MHC class II molecules may be important in its pathogenesis.

Triplet repeat expansion in myotonic dystrophy alters the adjacent chromatin structure.

Myotonic dystrophy is caused by an expansion of a CTG triplet repeat sequence in the 3' noncoding region of a protein kinase gene, yet the mechanism by which the triplet repeat expansion causes disease remains unknown. This report demonstrates that a DNase I hypersensitive site is positioned 3' of the triplet repeat in the wild-type allele in both fibroblasts and skeletal muscle cells. In three unrelated individuals with myotonic dystrophy that have large expansions of the triplet repeat, the allele with the triplet repeat expansion exhibited both overall DNase I resistance and inaccessibility of nucleases to the adjacent hypersensitive site. These results indicate that the triplet repeat expansion alters the adjacent chromatin structure, establishing a region of condensed chromatin, and suggests a molecular mechanism for myotonic dystrophy.

Análise de frequências alélicas de 15 marcadores STR em alunos da Faculdade de Odontologia de Bauru

Entre as muitas aplicações das tecnologias de identificação biológica humana, estão as finalidades forenses. O objetivo desta pesquisa foi verificar frequências alélicas de Short Tandem Repeat (STR) e os parâmetros estatísticos de interesse em genética de populações e forense para desenvolver o primeiro banco de dados populacional de DNA na Faculdade de Odontologia de Bauru, Universidade de São Paulo, (FOB/USP) para futuros usos forenses. Frequências alélicas de 15 locos autossômicos e do marcador de gênero amelogenina foram determinadas utilizando amostras de 200 μL de saliva doados por 296 alunos de graduação da FOB/USP, com idade ≥ 18 anos, após aprovação ética. Os testes laboratoriais foram feitos com kits comerciais. Resultados e parâmetros estatísticos foram obtidos por meio de programas clássicos: GeneMapper-ID-X, MS Excel 2002 versão 10.6871.6870, GenAlEx 6.5 e Arlequin 3.5, comparando quatro populações (brasileira, portuguesa, norte-americana e a população deste estudo). Os locos mais polimórficos foram D18S51 (17 alelos) e FGA (15 alelos), seguidos pelo D21S11 (13 alelos) e os menos polimórficos foram D16S539 e TH01 (7 alelos cada). A análise comparativa com amostra da população brasileira proveniente de estudos anteriores (n > 100.000) pelo teste goodness of fit X2 index não mostrou diferenças significativas entre estes grupos (p = 0,9999). Outros parâmetros estatísticos foram calculados comparando as populações: local (deste estudo), portuguesa e norte-americana. A análise de variância molecular (AMOVA) entre as três populações, entre as pessoas da mesma população e para cada pessoa de cada população mostrou que existe uma elevada variância individual (99%), que esta variância é mantida uniformemente entre as pessoas da mesma amostra/região (1%) e entre as três populações estudadas (0%). O estudo confirmou o elevado grau de polimorfismo e a alta heterozigosidade (96,5%) da população. Houve diferença significativa quanto ao gênero (79,7% mulheres) quando comparado à população brasileira em geral (50,4%), explicada pelas características do corpo discente da FOB/USP composto por 80,6% de pessoas do gênero feminino. Interessante foi a observação de uma microvariante alélica no loco D18S51, fora da escada padrão e da escala de abrangência do kit, correspondente ao alelo 29, ainda não definida na base de dados internacional (STRBase, atualizada em 07/08/2015). Esta microvariante deverá ser confirmada por testes familiares e sequenciamento de DNA para verificar a possibilidade de outra ocorrência familiar ou duplicação de nucleotídeos. No futuro, os dados obtidos neste estudo devem ser incorporados ao banco de dados da população brasileira e podem ser considerados como referência genética da população regional, ajudando a elucidar casos forenses. Após a confirmação, a potencial nova microvariante alélica contribuirá para a base de dados internacional STRBase.

Polymorphic CAG repeat length in the androgen receptor gene and association with neurodegeneration in a heterozygous female carrier of Kennedy's disease

Kennedy's disease (spinobulbar muscular atrophy) is an X-linked form of motor neuron disease affecting adult males carrying a CAG trinucleotide repeat expansion within the androgen receptor gene. While expression of Kennedy's disease is thought to be confined to males carrying the causative mutation, subclinical manifestations have been reported in a few female carriers of the disease. The reasons that females are protected from the disease are not clear, especially given that all other diseases caused by CAG expansions display dominant expression. In the current study, we report the identification of a heterozygote female carrying the Kennedy's disease mutation who was clinically diagnosed with motor neuron disease. We describe analysis of CAG repeat number in this individual as well as 33 relatives within the pedigree, including two male carriers of the Kennedy's mutation. The female heterozygote carried one expanded allele of the androgen receptor gene with CAG repeats numbering in the Kennedy's disease range (44 CAGs), with the normal allele numbering in the upper-normal range (28 CAGs). The subject has two sons, one of whom carries the mutant allele of the gene and has been clinically diagnosed with Kennedy's disease, whilst the other son carries the second allele of the gene with CAGs numbering in the upper normal range and displays a normal phenotype. This coexistence of motor neuron disease and the presence of one expanded allele and one allele at the upper limit of the normal range may be a coincidence. However, we hypothesize that the expression of the Kennedy's disease mutation combined with a second allele with a large but normal CAG repeat sequence may have contributed to the motor neuron degeneration displayed in the heterozygote female and discuss the possible reasons for phenotypic expression in particular individuals.