The frequency of CD127(low) expressing CD4(+)CD25(high) T regulatory cells is inversely correlated with human T lymphotrophic virus type-1 (HTLV-1) proviral load in HTLV-1-infection and HTLV-1-associated myelopathy/tropical spastic paraparesis

Background: CD4(+)CD25(high) regulatory T (T(Reg)) cells modulate antigen-specific T cell responses, and can suppress anti-viral immunity. In HTLV-1 infection, a selective decrease in the function of T(Reg) cell mediated HTLV-1-tax inhibition of FOXP3 expression has been described. The purpose of this study was to assess the frequency and phenotype of T(Reg) cells in HTLV-1 asymptomatic carriers and in HTLV-1-associated neurological disease (HAM/TSP) patients, and to correlate with measures of T cell activation. Results: We were able to confirm that HTLV-1 drives activation, spontaneous IFN gamma production, and proliferation of CD4+ T cells. We also observed a significantly lower proportion of CTLA-4(+) T(Reg) cells (CD4(+)CD25(high) T cells) in subjects with HAM/TSP patients compared to healthy controls. Ki-67 expression was negatively correlated to the frequency of CTLA-4(+) T(Reg) cells in HAM/TSP only, although Ki-67 expression was inversely correlated with the percentage of CD127(low) T(Reg) cells in healthy control subjects. Finally, the proportion of CD127(low) T(Reg) cells correlated inversely with HTLV-1 proviral load. Conclusion: Taken together, the results suggest that T(Reg) cells may be subverted in HAM/TSP patients, which could explain the marked cellular activation, spontaneous cytokine production, and proliferation of CD4(+) T cells, in particular those expressing the CD25(high)CD127(low) phenotype. T(Reg) cells represent a potential target for therapeutic intervention for patients with HTLV-1-related neurological diseases.

The C242T polymorphism of the p22-phox gene (CYBA) is associated with higher left ventricular mass in Brazilian hypertensive patients

Background: Reactive oxygen species have been implicated in the physiopathogenesis of hypertensive end-organ damage. This study investigated the impact of the C242T polymorphism of the p22-phox gene (CYBA) on left ventricular structure in Brazilian hypertensive subjects. Methods: We cross-sectionally evaluated 561 patients from 2 independent centers [Campinas (n = 441) and Vitoria (n = 120)] by clinical history, physical examination, anthropometry, analysis of metabolic and echocardiography parameters as well as p22-phox C242T polymorphism genotyping. In addition, NADPH-oxidase activity was quantified in peripheral mononuclear cells from a subgroup of Campinas sample. Results: Genotype frequencies in both samples were consistent with the Hardy-Weinberg equilibrium. Subjects with the T allele presented higher left ventricular mass/height(2.7) than those carrying the CC genotype in Campinas (76.8 +/- 1.6 vs 70.9 +/- 1.4 g/m(2.7); p = 0.009), and in Vitoria (45.6 +/- 1.9 vs 39.9 +/- 1.4 g/m(2.7); p = 0.023) samples. These results were confirmed by stepwise regression analyses adjusted for age, gender, blood pressure, metabolic variables and use of anti-hypertensive medications. In addition, increased NADPH-oxidase activity was detected in peripheral mononuclear cells from T allele carriers compared with CC genotype carriers (p = 0.03). Conclusions: The T allele of the p22-phox C242T polymorphism is associated with higher left ventricular mass/height(2.7) and increased NADPH-oxidase activity in Brazilian hypertensive patients. These data suggest that genetic variation within NADPH-oxidase components may modulate left ventricular remodeling in subjects with systemic hypertension.

Factor (NF) kappa B polymorphism is associated with heart function in patients with heart failure

Background: Cardiac remodeling is generally an adverse sign and is associated with heart failure (HF) progression. NFkB, an important transcription factor involved in many cell survival pathways, has been implicated in the remodeling process, but its role in the heart is still controversial. Recently, a promoter polymorphism associated with a lesser activation of the NFKB1 gene was also associated with Dilated Cardiomyopathy. The purpose of this study was to evaluate the association of this polymorphism with clinical and functional characteristics of heart failure patients of different etiologies. Methods: A total of 493 patients with HF and 916 individuals from a cohort of individuals from the general population were investigated. The NFKB1-94 insertion/deletion ATTG polymorphism was genotyped by High Resolution Melt discrimination. Allele and genotype frequencies were compared between groups. In addition, frequencies or mean values of different phenotypes associated with cardiovascular disease were compared between genotype groups. Finally, patients were prospectively followed-up for death incidence and genotypes for the polymorphism were compared regarding disease onset and mortality incidence in HF patients. Results: We did not find differences in genotype and allelic frequencies between cases and controls. Interestingly, we found an association between the ATTG(1)/ATTG(1) genotype with right ventricle diameter (P = 0.001), left ventricle diastolic diameter (P = 0.04), and ejection fraction (EF) (P = 0.016), being the genotype ATTG(1)/ATTG(1) more frequent in patients with EF lower than 50% (P = 0.01). Finally, we observed a significantly earlier disease onset in ATTG(1)/ATTG(1) carriers. Conclusion: There is no genotype or allelic association between the studied polymorphism and the occurrence of HF in the tested population. However, our data suggest that a diminished activation of NFKB1, previously associated with the ATTG(1)/ATTG(1) genotype, may act modulating on the onset of disease and, once the individual has HF, the genotype may modulate disease severity by increasing cardiac remodeling and function deterioration.

Association of alpha1a-adrenergic receptor polymorphism and blood pressure phenotypes in the Brazilian population

Background: The alpha1A-adrenergic receptor (alpha(1A)-AR) regulates the cardiac and peripheral vascular system through sympathetic activation. Due to its important role in the regulation of vascular tone and blood pressure, we aimed to investigate the association between the Arg347Cys polymorphism in the alpha(1A)-AR gene and blood pressure phenotypes, in a large sample of Brazilians from an urban population. Methods: A total of 1568 individuals were randomly selected from the general population of the Vitoria City metropolitan area. Genetic analysis of the Arg347Cys polymorphism was conducted by polymerase chain reaction/restriction fragment length polymorphism. We have compared cardiovascular risk variables and genotypes using ANOVA, and Chi-square test for univariate comparisons and logistic regression for multivariate comparisons. Results: Association analysis indicated a significant difference between genotype groups with respect to diastolic blood pressure (p = 0.04), but not systolic blood pressure (p = 0.12). In addition, presence of the Cys/Cys genotype was marginally associated with hypertension in our population (p = 0.06). Significant interaction effects were observed between the studied genetic variant, age and physical activity. Presence of the Cys/Cys genotype was associated with hypertension only in individuals with regular physical activity (odds ratio = 1.86; p = 0.03) or younger than 45 years (odds ratio = 1.27; p = 0.04). Conclusion: Physical activity and age may potentially play a role by disclosing the effects of the Cys allele on blood pressure. According to our data it is possible that the Arg347Cys polymorphism can be used as a biomarker to disease risk in a selected group of individuals.

TCF7L2 variant genotypes and type 2 diabetes risk in Brazil: significant association, but not a significant tool for risk stratification in the general population

Background: Genetic polymorphisms of the TCF7L2 gene are strongly associated with large increments in type 2 diabetes risk in different populations worldwide. In this study, we aimed to confirm the effect of the TCF7L2 polymorphism rs7903146 on diabetes risk in a Brazilian population and to assess the use of this genetic marker in improving diabetes risk prediction in the general population. Methods: We genotyped the single nucleotide polymorphisms (SNP) rs7903146 of the TCF7L2 gene in 560 patients with known coronary disease enrolled in the MASS II (Medicine, Angioplasty, or Surgery Study) Trial and in 1,449 residents of Vitoria, in Southeast Brazil. The associations of this gene variant to diabetes risk and metabolic characteristics in these two different populations were analyzed. To access the potential benefit of using this marker for diabetes risk prediction in the general population we analyzed the impact of this genetic variant on a validated diabetes risk prediction tool based on clinical characteristics developed for the Brazilian general population. Results: SNP rs7903146 of the TCF7L2 gene was significantly associated with type 2 diabetes in the MASS-II population (OR = 1.57 per T allele, p = 0.0032), confirming, in the Brazilian population, previous reports of the literature. Addition of this polymorphism to an established clinical risk prediction score did not increased model accuracy (both area under ROC curve equal to 0.776). Conclusion: TCF7L2 rs7903146 T allele is associated with a 1.57 increased risk for type 2 diabetes in a Brazilian cohort of patients with known coronary heart disease. However, the inclusion of this polymorphism in a risk prediction tool developed for the general population resulted in no improvement of performance. This is the first study, to our knowledge, that has confirmed this recent association in a South American population and adds to the great consistency of this finding in studies around the world. Finally, confirming the biological association of a genetic marker does not guarantee improvement on already established screening tools based solely on demographic variables.

An empirical evaluation of imputation accuracy for association statistics reveals increased type-I error rates in genome-wide associations

Background: Genome wide association studies (GWAS) are becoming the approach of choice to identify genetic determinants of complex phenotypes and common diseases. The astonishing amount of generated data and the use of distinct genotyping platforms with variable genomic coverage are still analytical challenges. Imputation algorithms combine directly genotyped markers information with haplotypic structure for the population of interest for the inference of a badly genotyped or missing marker and are considered a near zero cost approach to allow the comparison and combination of data generated in different studies. Several reports stated that imputed markers have an overall acceptable accuracy but no published report has performed a pair wise comparison of imputed and empiric association statistics of a complete set of GWAS markers. Results: In this report we identified a total of 73 imputed markers that yielded a nominally statistically significant association at P < 10(-5) for type 2 Diabetes Mellitus and compared them with results obtained based on empirical allelic frequencies. Interestingly, despite their overall high correlation, association statistics based on imputed frequencies were discordant in 35 of the 73 (47%) associated markers, considerably inflating the type I error rate of imputed markers. We comprehensively tested several quality thresholds, the haplotypic structure underlying imputed markers and the use of flanking markers as predictors of inaccurate association statistics derived from imputed markers. Conclusions: Our results suggest that association statistics from imputed markers showing specific MAF (Minor Allele Frequencies) range, located in weak linkage disequilibrium blocks or strongly deviating from local patterns of association are prone to have inflated false positive association signals. The present study highlights the potential of imputation procedures and proposes simple procedures for selecting the best imputed markers for follow-up genotyping studies.

CYP2C19 and ABCB1 gene polymorphisms are differently distributed according to ethnicity in the Brazilian general population

Background: Recent studies have reported the clinical importance of CYP2C19 and ABCB1 polymorphisms in an individualized approach to clopidogrel treatment. The aims of this study were to evaluate the frequencies of CYP2C19 and ABCB1 polymorphisms and to identify the clopidogrel-predicted metabolic phenotypes according to ethnic groups in a sample of individuals representative of a highly admixtured population. Methods: One hundred and eighty-three Amerindians and 1,029 subjects of the general population of 4 regions of the country were included. Genotypes for the ABCB1c.C3435T (rs1045642), CYP2C19*2 (rs4244285), CYP2C19*3 (rs4986893), CYP2C19*4 (rs28399504), CYP2C19*5 (rs56337013), and CYP2C19*17 (rs12248560) polymorphisms were detected by polymerase chain reaction followed by high resolution melting analysis. The CYP2C19*3, CYP2C19*4 and CYP2C19*5 variants were genotyped in a subsample of subjects (300 samples randomly selected). Results: The CYP2C19*3 and CYP2C19*5 variant alleles were not detected and the CYP2C19*4 variant allele presented a frequency of 0.3%. The allelic frequencies for the ABCB1c.C3435T, CYP2C19*2 and CYP2C19*17 polymorphisms were differently distributed according to ethnicity: Amerindian (51.4%, 10.4%, 15.8%); Caucasian descent (43.2%, 16.9%, 18.0%); Mulatto (35.9%, 16.5%, 21.3%); and African descent (32.8%, 20.2%, 26.3%) individuals, respectively. As a result, self-referred ethnicity was able to predict significantly different clopidogrel-predicted metabolic phenotypes prevalence even for a highly admixtured population. Conclusion: Our findings indicate the existence of inter-ethnic differences in the ABCB1 and CYP2C19 variant allele frequencies in the Brazilian general population plus Amerindians. This information could help in stratifying individuals from this population regarding clopidogrel-predicted metabolic phenotypes and design more cost-effective programs towards individualization of clopidogrel therapy.

Hypotheses, rationale, design, and methods for prognostic evaluation in type 2 diabetic patients with angiographically normal coronary arteries. The MASS IV-DM Trial

Background: The MASS IV-DM Trial is a large project from a single institution, the Heart Institute (InCor), University of Sao Paulo Medical School, Brazil to study ventricular function and coronary arteries in patients with type 2 diabetes mellitus. Methods/Design: The study will enroll 600 patients with type 2 diabetes who have angiographically normal ventricular function and coronary arteries. The goal of the MASS IV-DM Trial is to achieve a long-term evaluation of the development of coronary atherosclerosis by using angiograms and coronary-artery calcium scan by electron-beam computed tomography at baseline and after 5 years of follow-up. In addition, the incidence of major cardiovascular events, the dysfunction of various organs involved in this disease, particularly microalbuminuria and renal function, will be analyzed through clinical evaluation. In addition, an effort will be made to investigate in depth the presence of major cardiovascular risk factors, especially the biochemical profile, metabolic syndrome inflammatory activity, oxidative stress, endothelial function, prothrombotic factors, and profibrinolytic and platelet activity. An evaluation will be made of the polymorphism as a determinant of disease and its possible role in the genesis of micro- and macrovascular damage. Discussion: The MASS IV-DM trial is designed to include diabetic patients with clinically suspected myocardial ischemia in whom conventional angiography shows angiographically normal coronary arteries. The result of extensive investigation including angiographic follow-up by several methods, vascular reactivity, pro-thrombotic mechanisms, genetic and biochemical studies may facilitate the understanding of so-called micro- and macrovascular disease of DM.

Evaluating gene by sex and age interactions on cardiovascular risk factors in Brazilian families

Background: In family studies, it is important to evaluate the impact of genes and environmental factors on traits of interest. In particular, the relative influences of both genes and the environment may vary in different strata of the population of interest, such as young and old individuals, or males and females. Methods: In this paper, extensions of the variance components model are used to evaluate heterogeneity in the genetic and environmental variance components due to the effects of sex and age (the cutoff between young and old was 43 yrs). The data analyzed were from 81 Brazilian families (1,675 individuals) of the Baependi Family Heart Study. Results: The models allowing for heterogeneity of variance components by sex suggest that genetic and environmental variances are not different in males and females for diastolic blood pressure, LDL-cholesterol, and HDL-cholesterol, independent of the covariates included in the models. However, for systolic blood pressure, fasting glucose and triglycerides, the evidence for heterogeneity was dependent on the covariates in the model. For instance, in the presence of sex and age covariates, heterogeneity in the genetic variance component was suggested for fasting glucose. But, for systolic blood pressure, there was no evidence of heterogeneity in any of the two variance components. Except for the LDL-cholesterol, models allowing for heterogeneity by age provide evidence of heterogeneity in genetic variance for triglycerides and systolic and diastolic blood pressure. There was evidence of heterogeneity in environmental variance in fasting glucose and HDL-cholesterol. Conclusions: Our results suggest that heterogeneity in trait variances should not be ignored in the design and analyses of gene-finding studies involving these traits, as it may generate additional information about gene effects, and allow the investigation of more sophisticated models such as the model including sex-specific oligogenic variance components.

A comparison of lower limb EMG and ground reaction forces between barefoot and shod gait in participants with diabetic neuropathic and healthy controls

Background: It is known that when barefoot, gait biomechanics of diabetic neuropathic patients differ from nondiabetic individuals. However, it is still unknown whether these biomechanical changes are also present during shod gait which is clinically advised for these patients. This study investigated the effect of the participants own shoes on gait biomechanics in diabetic neuropathic individuals compared to barefoot gait patterns and healthy controls. Methods: Ground reaction forces and lower limb EMG activities were analyzed in 21 non-diabetic adults (50.9 +/- 7.3 yr, 24.3 +/- 2.6 kg/m(2)) and 24 diabetic neuropathic participants (55.2 +/- 7.9 yr, 27.0 +/- 4.4 kg/m(2)). EMG patterns of vastus lateralis, lateral gastrocnemius and tibialis anterior, along with the vertical and antero-posterior ground reaction forces were studied during shod and barefoot gait. Results: Regardless of the disease, walking with shoes promoted an increase in the first peak vertical force and the peak horizontal propulsive force. Diabetic individuals had a delay in the lateral gastrocnemius EMG activity with no delay in the vastus lateralis. They also demonstrated a higher peak horizontal braking force walking with shoes compared to barefoot. Diabetic participants also had a smaller second peak vertical force in shod gait and a delay in the vastus lateralis EMG activity in barefoot gait compared to controls. Conclusions: The change in plantar sensory information that occurs when wearing shoes revealed a different motor strategy in diabetic individuals. Walking with shoes did not attenuate vertical forces in either group. Though changes in motor strategy were apparent, the biomechanical did not support the argument that the use of shoes contributes to altered motor responses during gait.

Efficacy of a 7-day course of furazolidone, levofloxacin, and lansoprazole after failed Helicobacter pylori eradication

Background: Increasing resistance to clarithromycin and nitroimidazole is the main cause of failure in the Helicobacter pylori eradication. The ideal retreatment regimen remains unclear, especially in developing countries, where the infection presents high prevalence and resistance to antibiotics. The study aimed at determining the efficacy, compliance and adverse effects of a regimen that included furazolidone, levofloxacin and lansoprazole in patients with persistent Helicobacter pylori infection, who had failed to respond to at least one prior eradication treatment regimen. Methods: This study included 48 patients with peptic ulcer disease. Helicobacter pylori infection was confirmed by a rapid urease test and histological examination of samples obtained from the antrum and corpus during endoscopy. The eradication therapy consisted of a 7-day twice daily oral administration of lansoprazole 30 mg, furazolidone 200 mg and levofloxacin 250 mg. Therapeutic success was confirmed by a negative rapid urease test, histological examination and 14C- urea breath test, performed 12 weeks after treatment completion. The Chi-square method was used for comparisons among eradication rates, previous treatments and previous furazolidone use. Results: Only one of the 48 patients failed to take all medications, which was due to adverse effects (vomiting). Per-protocol and intention-to-treat eradication rates were 89% (95% CI-89%-99%) and 88% (88-92%), respectively. Mild and moderate adverse effects were reported by 41 patients (85%). For patients with one previous treatment failure, the eradication rate was 100%. Compared to furazolidone-nave patients, eradication rates were lower in those who had failed prior furazolidone-containing regimen(s) (74% vs. 100%, p = 0.002). Conclusion: An empiric salvage-regimen including levofloxacin, furazolidone and lansoprazole is very effective in the eradication of Helicobacter pylori, particularly in patients that have failed one prior eradication therapy.

249 TP53 mutation has high prevalence and is correlated with larger and poorly differentiated HCC in Brazilian patients

Background: Ser-249 TP53 mutation (249(Ser)) is a molecular evidence for aflatoxin-related carcinogenesis in Hepatocellular Carcinoma (HCC) and it is frequent in some African and Asian regions, but it is unusual in Western countries. HBV has been claimed to add a synergic effect on genesis of this particular mutation with aflatoxin. The aim of this study was to investigate the frequency of 249Ser mutation in HCC from patients in Brazil. Methods: We studied 74 HCC formalin fixed paraffin blocks samples of patients whom underwent surgical resection in Brazil. 249Ser mutation was analyzed by RFLP and DNA sequencing. HBV DNA presence was determined by Real-Time PCR. Results: 249Ser mutation was found in 21/74 (28%) samples while HBV DNA was detected in 13/74 (16%). 249Ser mutation was detected in 21/74 samples by RFLP assay, of which 14 were confirmed by 249Ser mutant-specific PCR, and 12 by nucleic acid sequencing. All HCC cases with p53-249ser mutation displayed also wild-type p53 sequences. Poorly differentiated HCC was more likely to have 249Ser mutation (OR = 2.415, 95% CI = 1.001 - 5.824, p = 0.05). The mean size of 249Ser HCC tumor was 9.4 cm versus 5.5 cm on wild type HCC (p = 0.012). HBV DNA detection was not related to 249Ser mutation. Conclusion: Our results indicate that 249Ser mutation is a HCC important factor of carcinogenesis in Brazil and it is associated to large and poorly differentiated tumors.

Evaluation of muscle strength and motor abilities in children with type II and III spinal muscle atrophy treated with valproic acid

Background: Spinal muscular atrophy (SMA) is an autosomal recessive disorder that affects the motoneurons of the spinal anterior horn, resulting in hypotonia and muscle weakness. The disease is caused by deletion or mutation in the telomeric copy of SMN gene (SMN1) and clinical severity is in part determined by the copy number of the centromeric copy of the SMN gene (SMN2). The SMN2 mRNA lacks exon 7, resulting in a production of lower amounts of the full-length SMN protein. Knowledge of the molecular mechanism of diseases has led to the discovery of drugs capable of increasing SMN protein level through activation of SMN2 gene. One of these drugs is the valproic acid (VPA), a histone deacetylase inhibitor. Methods: Twenty-two patients with type II and III SMA, aged between 2 and 18 years, were treated with VPA and were evaluated five times during a one-year period using the Manual Muscle Test (Medical Research Council scale-MRC), the Hammersmith Functional Motor Scale (HFMS), and the Barthel Index. Results: After 12 months of therapy, the patients did not gain muscle strength. The group of children with SMA type II presented a significant gain in HFMS scores during the treatment. This improvement was not observed in the group of type III patients. The analysis of the HFMS scores during the treatment period in the groups of patients younger and older than 6 years of age did not show any significant result. There was an improvement of the daily activities at the end of the VPA treatment period. Conclusion: Treatment of SMA patients with VPA may be a potential alternative to alleviate the progression of the disease.

High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load

Background: Concomitant infections may influence HIV progression by causing chronic activation leading to decline in T-cell function. In the Americas, visceral (AVL) and tegumentary leishmaniasis (ATL) have emerged as important opportunistic infections in HIV-AIDS patients and both of those diseases have been implicated as potentially important co-factors in disease progression. We investigated whether leishmaniasis increases lymphocyte activation in HIV-1 co-infected patients. This might contribute to impaired cellular immune function. Methods: To address this issue we analyzed CD4(+) T absolute counts and the proportion of CD8(+) T cells expressing CD38 in Leishmania/HIV co-infected patients that recovered after anti-leishmanial therapy. Results: We found that, despite clinical remission of leishmaniasis, AVL co-infected patients presented a more severe immunossupression as suggested by CD4(+) T cell counts under 200 cells/mm(3), differing from ATL/HIV-AIDS cases that tends to show higher lymphocytes levels (over 350 cells/mm(3)). Furthermore, five out of nine, AVL/HIV-AIDS presented low CD4(+) T cell counts in spite of low or undetectable viral load. Expression of CD38 on CD8(+) T lymphocytes was significantly higher in AVL or ATL/HIV-AIDS cases compared to HIV/AIDS patients without leishmaniasis or healthy subjects. Conclusions: Leishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4(+) T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.

Proteome analysis of schizophrenia patients Wernicke's area reveals an energy metabolism dysregulation

Background: Schizophrenia is likely to be a consequence of DNA alterations that, together with environmental factors, will lead to protein expression differences and the ultimate establishment of the illness. The superior temporal gyrus is implicated in schizophrenia and executes functions such as the processing of speech, language skills and sound processing. Methods: We performed an individual comparative proteome analysis using two-dimensional gel electrophoresis of 9 schizophrenia and 6 healthy control patients' left posterior superior temporal gyrus (Wernicke's area - BA22p) identifying by mass spectrometry several protein expression alterations that could be related to the disease. Results: Our analysis revealed 11 downregulated and 14 upregulated proteins, most of them related to energy metabolism. Whereas many of the identified proteins have been previously implicated in schizophrenia, such as fructose-bisphosphate aldolase C, creatine kinase and neuron-specific enolase, new putative disease markers were also identified such as dihydrolipoyl dehydrogenase, tropomyosin 3, breast cancer metastasis-suppressor 1, heterogeneous nuclear ribonucleoproteins C1/C2 and phosphate carrier protein, mitochondrial precursor. Besides, the differential expression of peroxiredoxin 6 (PRDX6) and glial fibrillary acidic protein (GFAP) were confirmed by western blot in schizophrenia prefrontal cortex. Conclusion: Our data supports a dysregulation of energy metabolism in schizophrenia as well as suggests new markers that may contribute to a better understanding of this complex disease.