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BACKGROUND Mean arterial pressure (MAP) drives ocular perfusion. Excessive 24-h MAP variability relates to glaucoma, however, whether this is due to dips or increases in the blood pressure (BP) is undocumented. We investigated the association of open-angle glaucoma (OAG) in relation to the 5 largest MAP dips/increases over 24-h, henceforth called dips/blips. METHODS In the Maracaibo Aging Study (MAS), 93 participants aged ≥40 y (women, 87.1%; mean age, 61.9 y) underwent baseline ophthalmological and 24-h ambulatory BP monitoring assessments. OAG was the presence of optic nerve damage and visual field defects. Statistical methods included logistic regression and the generalized R2 statistic. For replication, 48 OAG cases at the Leuven Glaucoma Clinic were matched with 48 controls recruited from Flemish population. RESULTS In the MAS, 26 participants had OAG. OAG compared to non-OAG participants experienced longer and deeper dips (116.5 vs. 102.7 minutes; to 60.3 vs. 66.6 mm Hg; −21.0 vs. −18.0 mm Hg absolute or 0.79 vs. 0.81 relative dip compared to the preceding reading). The adjusted odds ratios associated with dip measures ranged from 2.25 (95% confidence interval [CI], 1.31–4.85; P = 0.009) to 3.39 (95% CI, 1.36–8.46; P = 0.008). On top of covariables and 24-MAP level/variability, the dip measures increased the model performance (P ≤ 0.025). Blips did not associate with OAG. The case–control study replicated the MAS observations. CONCLUSIONS Dips rather than increases in the 24-h MAP level were associated with increased risk for OAG. An ophthalmological examination combined with 24-h BP monitoring might be precautious steps required in normotensive and hypertensive patients at risk of OAG.

Background Venezuela is in the throes of a complex humanitarian crisis that is one of the worst in decades to impact any country outside of wartime. This case analysis describes the challenges faced by the ongoing Maracaibo Aging Study (MAS) during the deteriorating conditions in Venezuela. When the MAS began in 1997, it focused on memory-related disorders. Since then, strategic planning and proactive community participation allowed us to anticipate and address logistical, funding, and ethical challenges, and facilitated the enrollment and retention of more than 2500 subjects over 55 years of age. All participants, who are residents of the city of Maracaibo, Venezuela, underwent various assessments on several occasions. Here, we discuss how our approach to implementing a longitudinal, population-based study of age-related conditions has allowed our research program to continue throughout this period of political, economic, and social upheaval. Discussion As the social context in Venezuela became more complicated, new challenges emerged, and strategies to sustain the study and participation were refined. We identified five main mechanisms through which the evolving humanitarian crisis has affected implementation of the MAS: 1) community dynamics; 2) morale of researchers, staff, and participants; 3) financial feasibility; 4) components of the research process; and 5) impact on the health of staff, participants, and their families. Strategies to compensate for the impact on these components were implemented, based on inputs from community members and staff. Improved communication, greater involvement of stakeholders, broadening the scope of the project, and strengthening international collaboration have been the most useful strategies. Particular demands emerged, related to the increased mortality and comorbidities of participants and staff, and deterioration of basic services and safety. Conclusion Although the MAS has faced numerous obstacles, it has been possible to continue a longitudinal research project throughout the humanitarian crisis, because our research team has engaged the community deeply and developed a sense of mutual commitment, and also because our project has provided funding to help keep researchers employed, somewhat attenuating the brain drain.

Hypoperfusion of the optic nerve might be involved in the pathogenesis of normal-tension glaucomatous optic neuropathy (GON). Mean arterial pressure (MAP) drives ocular perfusion, but no previous studies have addressed the risk of GON in relation to blood pressure (BP) variability, independent of BP level. In a cross-sectional study, 93 residents of Maracaibo, Venezuela, underwent optical coherence tomography, visual field assessments and 24-h ambulatory BP monitoring between 2011 and 2016. We investigated the association of normal-tension GON with or without visual field defects with reading-to reading variability of 24-h MAP, as captured by variability independent of the MAP level (VIMmap). Odds ratios (ORs) were adjusted for 24-h MAP level and for a propensity score of up to five risk factors. Among the 93 participants (87.1% women; mean age, 61.9 years), 26 had open-angle normal-tension GON at both eyes; 14 had visual field defects; and 19 did not have visual field defects. The OR ratios for normal-tension GON, expressed per 1-SD increment in VIMmap (2 mm Hg), were 2.17 (95% confidence interval, 1.33-3.53) unadjusted; 2.20 (1.35-3.61) adjusted for 24-h MAP level only; 1.93 (1.10-3.41) with additional adjustment for age, educational attainment, high-density lipoprotein (HDL) cholesterol and office hypertension; and 1.95 (1.10-3.45) in models including intraocular pressure. We confirmed our a priori hypothesis that BP variability, most likely operating via hypoperfusion of the optic nerve, is associated with normal-tension GON. 24-H ambulatory BP monitoring might therefore help stratify the risk of normal-tension GON.

The accumulation of fat, especially in visceral sites, is a significant risk factor for several chronic diseases with altered cardiometabolic homeostasis. We studied how intensive long-term weight loss and subsequent weight regain affect physiological changes, by longitudinally interrogating the lipid metabolism and white blood cell transcriptomic markers in healthy, normal-weight individuals. The current study examined 42 healthy, young (age: 27.5 ± 4.0 years), normal-weight (body mass index, BMI: 23.4 ± 1.7 kg/m2) female athletes, of which 25 belong to the weight loss and regain group (diet group), and 17 to the control group. Participants were evaluated, and fasting blood samples were drawn at three time points: at baseline (PRE); at the end of the weight loss period (MID: 21.1 ± 3.1 weeks after PRE); and at the end of the weight regain period (POST: 18.4 ± 2.9 weeks after MID). Following the weight loss period, the diet group experienced a ~73% reduction (~0.69 kg) in visceral fat mass (false discovery rate, FDR −16), accompanied by anti-atherogenic effects on transcriptomic markers, decreased low-grade inflammation (e.g., as α1–acid glycoprotein (FDR = 3.08 × 10−13) and hs-CRP (FDR = 2.44 × 10−3)), and an increase in functionally important anti-atherogenic high-density lipoprotein -associated metabolites (FDR

Objectives Kazakhstan is a Central Asian crossroad of European and Asian populations situated along the way of the Great Silk Way. The territory of Kazakhstan has historically been inhabited by nomadic tribes and today is the multi-ethnic country with the dominant Kazakh ethnic group. We sequenced and analyzed the whole-genomes of five ethnic healthy Kazakh individuals with high coverage using next-generation sequencing platform. This whole-genome sequence data of healthy Kazakh individuals can be a valuable reference for biomedical studies investigating disease associations and population-wide genomic studies of ethnically diverse Central Asian region. Data description Blood samples have been collected from five ethnic healthy Kazakh individuals living in Kazakhstan. The genomic DNA was extracted from blood and sequenced. Sequencing was performed on Illumina HiSeq2000 next-generation sequencing platform. We sequenced and analyzed the whole-genomes of ethnic Kazakh individuals with the coverage ranging from 26 to 32X. Ranging from 98.85 to 99.58% base pairs were totally mapped and aligned on the human reference genome GRCh37 hg19. Het/Hom and Ts/Tv ratios for each whole genome ranged from 1.35 to 1.49 and from 2.07 to 2.08, respectively. Sequencing data are available in the National Center for Biotechnology Information SRA database under the accession number PRJNA374772.

Background: Very few studies have investigated the association between total plasma homocysteine (tHcy) and depressive symptoms in older Hispanics. Objective: To test the hypothesis that high tHcy associates with depressive symptoms in older Hispanics. Methods: A total of 1,418 participants .55 years old from the Maracaibo Aging Study (MAS) underwent standardized neurological, neuropsychiatric, and cardiovascular assessments. The Neuropsychiatric Inventory Depression Subscale (NPId) was used to assess the burden of depressive symptoms. The tHcy levels and other biochemical parameters in blood samples were measured. Univariate and multivariate logistic regression models were applied. Results: Participants with depressive symptoms had higher levels of tHcy than those without (15.1 versus 13.9 µmol/L; p = 0.009). Elevated tHcy levels were associated with depressive symptoms after adjusting for age, sex, education, smoking, diabetes, hypertension, alcohol intake, stroke, and dementia (OR = 1.58; 95% CI, 1.18–2.12). Conclusion: Elevated levels of tHcy were associated with depressive symptoms in older Hispanics living under the nutritional and environmental conditions of a developing country.

Although there is a growing interest in improving cognitive abilities in older adults, there has been considerable variation in how different cognitive domains have been conceptualized over time. Broadly, neuropsychology assumes that cognitive processes are fundamentally similar across humankind (Nell, 1999), and as such, the manner in which cognitive processes are assessed is similar across cultures (Rivera Mindt, Byrd, Saez, & Manly, 2010).

Background: Dementia of the Alzheimer’s type (DAT) impacts Hispanics disproportionately, with almost a twofold elevated risk of developing DAT, as well as earlier onset of the disease, than in non-Hispanic Whites. However, the role of main risk factors for DAT, such as APOE-ɛ4 and blood pressure (BP) levels, remains uncertain among Hispanics. Objective: To investigate the association of APOE-ɛ4 and BP levels, measures with 24-h ambulatory BP monitoring, with incidence of DAT in an elderly cohort of Hispanics. Methods: 1,320 participants from the Maracaibo Aging Study, free of dementia at the baseline, and with ambulatory BP measurements and APOE genotype available were included. Adjusted Cox proportional models were performed to examine 1) the incidence of DAT and 2) the relationship between BP levels and DAT according to APOE genotypes. Models were adjusted by competing risk of death before the onset of DAT. Model performance was assessed by likelihood test. Results: The average follow-up time was 5.3 years. DAT incidence was 5.8 per 1000 person-year. APOE-ɛ4 carriers had a higher risk of DAT. In unadjusted analyses, conventional, 24-h, and nighttime systolic BP levels were significantly higher in participants who developed DAT and of APOE-ɛ4 carriers (p < 0.05). After adjustment for competing risks, only higher nighttime systolic BP was associated with DAT incidence, but only among subjects carrying APOE-ɛ4. Conclusion: In this Hispanic population, both APOE-ɛ4 genotype and assessment of nocturnal systolic BP (rather than diurnal or office BP) were necessary to estimate DAT risk.

Hypoperfusion of the optic nerve might be involved in the pathogenesis of normal-tension glaucomatous optic neuropathy (GON). Mean arterial pressure (MAP) drives ocular perfusion, but no previous studies have addressed the risk of GON in relation to blood pressure (BP) variability, independent of BP level. In a cross-sectional study, 93 residents of Maracaibo, Venezuela, underwent optical coherence tomography, visual field assessments and 24-h ambulatory BP monitoring between 2011 and 2016. We investigated the association of normal-tension GON with or without visual field defects with reading-to reading variability of 24-h MAP, as captured by variability independent of the MAP level (VIM

Purpose To determine which nocturnal blood pressure (BP) parameters (low levels or extreme dipper status) are associated with an increased risk of glaucomatous damage in Hispanics. Design Observational cross-sectional study. Participants A subset (n = 93) of the participants from the Maracaibo Aging Study (MAS) who met the study eligibility criteria were included. These participants, who were at least 40 years of age, had measurements for optical tomography coherence, visual field (VF) tests, 24-hour BP, office BP, and intraocular pressure Methods Univariate and multivariate logistic regression analyses under the generalized estimating equations (GEE) framework were used to examine the relationships between glaucomatous damage and BP parameters, with particular attention to decreases in nocturnal BP. Main Outcome Measures Glaucomatous optic neuropathy (GON) based on the presence of optic nerve damage and VF defects. Results The mean age was 61.9 years, and 87.1% were women. Of 185 eyes evaluated, 19 (26.5%) had signs of GON. Individuals with GON had significantly lower 24-hour and nighttime diastolic BP levels than those without. However, results of the multivariate GEE models indicated that the glaucomatous damage was not related to the average systolic or diastolic BP levels measured over 24 hours, daytime, or nighttime. In contrast, extreme decreases in nighttime systolic and diastolic BP (>20% compared with daytime BP) were significant risk factors for glaucomatous damage (odds ratio, 19.78 and 5.55, respectively). Conclusions In this population, the link between nocturnal BP and GON is determined by extreme dipping effects rather than low nocturnal BP levels alone. Further studies considering extreme decreases in nocturnal BP in individuals at high risk of glaucoma are warranted.

Hypoperfusion of the optic nerve might be involved in the pathogenesis of normal-tension glaucomatous optic neuropathy (GON). Mean arterial pressure (MAP) drives ocular perfusion, but no previous studies have addressed the risk of GON in relation to blood pressure (BP) variability, independent of BP level. In a cross-sectional study, 93 residents of Maracaibo, Venezuela, underwent optical coherence tomography, visual field assessments and 24-h ambulatory BP monitoring between 2011 and 2016. We investigated the association of normal-tension GON with or without visual field defects with reading-to reading variability of 24-h MAP, as captured by variability independent of the MAP level (VIMmap). Odds ratios (ORs) were adjusted for 24-h MAP level and for a propensity score of up to five risk factors. Among the 93 participants (87.1% women; mean age, 61.9 years), 26 had open-angle normal-tension GON at both eyes; 14 had visual field defects; and 19 did not have visual field defects. The OR ratios for normal-tension GON, expressed per 1-SD increment in VIMmap (2 mm Hg), were 2.17 (95% confidence interval, 1.33–3.53) unadjusted; 2.20 (1.35–3.61) adjusted for 24-h MAP level only; 1.93 (1.10–3.41) with additional adjustment for age, educational attainment, high-density lipoprotein (HDL) cholesterol and office hypertension; and 1.95 (1.10–3.45) in models including intraocular pressure. We confirmed our a priori hypothesis that BP variability, most likely operating via hypoperfusion of the optic nerve, is associated with normal-tension GON. 24-H ambulatory BP monitoring might therefore help stratify the risk of normal-tension GON.

Higher physical activity is associated with a reduced hazard for a plethora of diseases. It has remained unknown how the two primary physical activity-associated health effects, improved physical performance and change in body composition, independently modulate metabolic profiles toward a reduced risk for adverse outcomes. Here, we utilized a prospective cohort of 664 young men undergoing military service. We studied the metabolic associations of changes in muscle performance and body composition during military service (range 6–12 mo). We subsequently replicated our results for body composition change in 234 population-based samples with a 7-yr follow-up. We found that increased physical performance was associated with reduced very-low-density lipoprotein (VLDL)-related measures [change in VLDL cholesterol: beta = −0.135; 95% confidence interval (CI) = −0.217, −0.054, P = 1.2 × 10−3] and lower inflammation (change in glycoprotein acetyls: beta = −0.138, 95% CI = −0.217, −0.059, P = 6.5 × 10−4), independent of change in body composition. Lower body fat percentage, independent of change in muscle performance, was associated with metabolic changes including lower low-density lipoprotein (LDL) cholesterol measures (change in LDL cholesterol: beta = −0.193, 95% CI = −0.295, −0.090; P = 2.5 × 10−4), increased high-density lipoprotein (HDL) cholesterol measures (change in large HDL cholesterol: beta = 0.316, 95% CI = 0.205, 0.427; P = 3.7 × 10−8), and decreased concentrations of amino acids (change in leucine concentration: beta = −0.236, 95% CI = −0.341, −0.132; P = 1.0 × 10−5) that are type 2 diabetes biomarkers. Importantly, all body fat percentage associations were replicated in a general population-based cohort. Our findings indicate that improved muscle performance showed weaker associations on the metabolic profiles than change in body composition and reduction in body fat percentage reduces cardiometabolic risk mediated by atherogenic lipoprotein particles and branched-chain and aromatic amino acid concentrations.

International audience; In 2016, during a major Zika virus (ZIKV) outbreak in Maracaibo, Venezuela, a 49-year-old woman and an unrelated 4-year-old boy developed bilateral optic neuritis 2-3 weeks after presenting an acute febrile illness characterized by low-grade fever, rash and myalgia [1]. Both patients presented sudden, painless bilateral loss of vision with no corneal or uveal abnormalities. Fundoscopic examination revealed oedema of the optic nerve and optic disc pallor. Optical coherence tomography confirmed bilateral optic nerve head swelling in the case of the adult, but it was not carried out in the child. Automated perimetry performed in the adult revealed bilateral diffuse visual field loss. Magnetic resonance imaging of the brain in both cases was unremarkable. Both patients were diagnosed with bilateral optic neuritis of possible infectious or parainfectious origin. Differential diagnoses that were considered and subsequently discarded included arteritic and non-arteritic ischaemic optic neuropathy, and brain disorders such as multiple sclerosis and brain tumours. Both patients were seropositive for anti-ZIKV IgG and seronegative for anti-ZIKV IgM. In addition, both patients were positive for anti-dengue virus (DENV) IgG for all four DENV serotypes. Management included intravenous methylprednisolone for 3 days, followed by oral prednisolone for 11 days. Although the patients presented a modest improvement in their vision, they continued to have visual impairment after several months of follow-up [1]. DISCUSSION Correct Answer: 4. These complications can lead to permanent visual impairment. ZIKV is a mosquito-borne RNA virus belonging to the genus Flavivirus of the family Flaviviridae [2]. The classical QUESTION Which of the following statements is accurate about non-congenital severe ocular complications of Zika virus (ZIKV) infection? ANSWER OPTIONS 1. They are unique to ZIKV infection and readily distinguish-able from complications caused by other flaviviruses. 2. Serious ocular complications are related to the severity of the acute exanthematous illness. 3. The diagnosis can be conclusively established by detecting anti-Zika IgM and/or IgG in the patient's serum. 4. These complications can lead to permanent visual impairment. 5. There is specific treatment for ocular manifestations caused by ZIKV infection. This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. 1 clinical picture of ZIKV infection includes fever, exanthema, headache and conjunctivitis. The most common non-congenital, ocular manifestation of ZIKV infection is a self-limiting conjunctivitis. Serious ocular complications have been reported for other arboviruses, such as DENV [3-20], chikungunya virus [21-24], West Nile virus [25-39] and Rift Valley fever virus [9-11, 40] (Table 1). To date, there is no specific ocular lesion that is pathognomonic for ZIKV infection [41-45]. Non-congenital ocular complications are infrequent, but serious, consequences of ZIKV and other arboviral infections. The complications may appear at the end of the acute febrile illness, but more commonly occur within 2 weeks to 1 month after the onset of symptoms. There is no evidence to suggest that serious ocular complications correlate with the severity of the acute febrile illness. One study, however, found that the white cell count and serum albumin are significant predictors of ocular complications of DENV [46]. Serological testing for arboviral diseases should be performed in all patients with ocular complications and a recent history of acute febrile exanthematous infection, who live, or have travelled to, endemic regions. The presence of IgM to ZIKV strongly suggests that the ocular manifestation is associated with this virus. A causative aetiology, however, can only be established by documenting the presence of the virus in body fluids, either by cell culture or by PCR. It should be noted that other viruses, such as herpes simplex virus and human immunodeficiency virus, can also cause retinal damage and optic neuritis. Furthermore, as in the cases presented here, the diagnosis is complicated by cross-reactivity among flaviviruses, and by the co-circulation of arboviruses. Most patients with ocular complications of arboviral infections recover completely. Nevertheless, physicians should be aware that a small percentage of patients have permanent damage with long-life visual impairment. There is no specific or established treatment for optic neuritis caused by any arboviral infection. Systemic steroids may be used to reduce inflammation and resulting ischae-mia. Corticosteroids have been used in combination with acyclovir to treat chikungunya-associated optic neuritis, but efficacy has not been proven [47].

Introduction. Although the current Zika virus (ZIKV) epidemic is a major public health concern, most reports have focused on congenital ZIKV syndrome, its most devastating manifestation. Severe ocular complications associated with ZIKV infections and possible pathogenetic factors are rarely described. Here, we describe three Venezuelan patients who developed severe ocular manifestations following ZIKV infections. We also analyse their serological response to ZIKV and dengue virus (DENV). Case presentation. One adult with bilateral optic neuritis, a child of 4 years of age with retrobulbar uveitis and a newborn with bilateral congenital glaucoma had a recent history of an acute exanthematous infection consistent with ZIKV infection. The results of ELISA tests indicated that all patients were seropositive for ZIKV and four DENV serotypes. Conclusion. Patients with ZIKV infection can develop severe ocular complications. Anti-DENV antibodies from previous infections could play a role in the pathogenesis of these complications. Well-designed epidemiological studies are urgently needed to measure the risk of ZIKV ocular complications and confirm whether they are associated with the presence of anti-flaviviral antibodies.

BACKGROUND Orthostatic hypotension (OH) occurs when mechanisms regulating blood pressure (BP) levels after standing-up are altered. It is unclear how prevalence and risk factors for OH are different between sexes. We aimed to investigate sex differences in prevalence and risk factors for OH elderly individuals. METHODS We included 882 participants from Maracaibo Aging Study. OH was a sustained reduction of ≥20 mm Hg in systolic BP, ≥10 mm Hg in diastolic BP, or both, after 3 minutes of changing positions from supine to standing. Multivariable logistic regression models were used to examine the relationships among risk factors for OH in men and women considering interaction sex-term and stratified by sex. RESULTS The mean age was 66.7 ± 8.5 years, being similar by sex. Women and men 55–74 years had similar prevalence of OH+ (18.5% vs. 20.9%, respectively). After 75 years, the proportion of women with OH+ was lower than men (11% vs. 30%, respectively). Hypertension, specifically systolic BP ≥140 mm Hg, and high pulse pressure (PP) were related with OH+ accounted by interaction sex-term, while diastolic BP ≥90 mm Hg, antihypertensive treatment, body mass index (BMI), diabetes mellitus and age were not. Systolic BP ≥140 mm Hg increases the risk of OH only among women, while BMI showed an inverse association in both sexes. CONCLUSIONS Although the prevalence of OH is similar in both sexes, there are different risk factors associated by sex. Systolic BP ≥140 mm Hg was associated with increased risk of OH only with women while BMI was a protective factor for OH in men and women.

Introduction There are few longitudinal studies of dementia in developing countries. We used longitudinal data from the Maracaibo Aging Study to accurately determine the age- and sex-specific incidence of dementia in elderly Latin Americans. Methods The Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition, Text Revision) was used to diagnose dementia, which was classified as Alzheimer's disease, vascular dementia, or other. Age- and sex-specific incidence was estimated as the number of new cases of dementia divided by person-years (p-y) of follow-up. Results The incidence of all dementia diagnoses was 9.10 per 1000 p-y (95% confidence interval [CI] 7.13–11.44; 8026 total p-y), 5.18 for Alzheimer's disease (95% CI 3.72–7.03; 7916 total p-y), and 3.35 for vascular dementia (95% CI 2.19–4.91; 7757 total p-y). Discussion Among Maracaibo Aging Study participants younger than 65 years, the incidence of dementia was higher than that of US Whites. Among individuals older than 65 years, the incidence was comparable to the mean of previous incidence estimates for other populations worldwide.

Acrophobia, an abnormal fear of heights, is a specific phobia characterized as apprehension cued by the occurrence or anticipation of elevated spaces. It is considered a complex trait with onset influenced by both genetic and environmental factors. Identification of genetic risk variants would provide novel insight into the genetic basis of the fear of heights phenotype and contribute to the molecular-level understanding of its aetiology. Genetic isolates may facilitate identification of susceptibility alleles due to reduced genetic heterogeneity. We took advantage of an internal genetic isolate in Finland in which a distinct acrophobia phenotype appears to be segregating in pedigrees originally ascertained for schizophrenia. We conducted parametric, nonparametric, joint linkage and linkage disequilibrium analyses using a microsatellite marker panel, genotyped in families to search for chromosomal regions correlated with acrophobia. Our results implicated a few regions with suggestive evidence for linkage on chromosomes 4q28 (LOD = 2.17), 8q24 (LOD = 2.09) and 13q21-q22 (LOD = 2.22). We observed no risk haplotypes shared between different families. These results suggest that genetic predisposition to acrophobia in this genetic isolate is unlikely to be mediated by a small number of shared high-risk alleles, but rather has a complex genetic architecture.

Coronary heart disease (CHD) is a complex disorder constituting a major health problem in Western societies. To assess the genetic background of CHD, we performed a genomewide linkage scan in two study samples from the genetically isolated population of Finland. An initial study sample consisted of family material from the northeastern part of Finland, settled by a small number of founders approximately 300 years ago. A second study sample originated from the southwestern region of Finland, settled approximately 2,000 years ago. Families were ascertained through probands exhibiting premature CHD, defined as >50% stenosis of at least two coronary arteries at a young age, as verified by coronary angiography. Both study samples and the pooled data set provided evidence for linkage in two chromosomal regions. A region on chromosome 2q21.1-22 yielded two-point LOD scores of 3.2, 1.9, and 3.7, in the affected sib-pair (ASP) analyses of the northeastern, southwestern, and pooled study samples. The corresponding multipoint maximum-likelihood scores (MLSs) for these three study samples were 2.4, 1.3, and 3.0. In addition, a region on chromosome Xq23-26 resulted in two-point LOD scores of 1.9, 3.5, and 2.9 and in multipoint MLSs of 3.4, 3.1, and 2.5, respectively. In conclusion, this study identifies two loci likely to contribute to premature CHD: one on chromosome 2q21.1-22 and another on chromosome Xq23-26.

It has been shown that parametric analysis of linkage disequilibrium conditional on linkage using an overly deterministic model can be optimal for family-based association analysis. However, if one applies this strategy carelessly, there is a risk of false inference. We analyse properties of such likelihood ratio tests when the assumed disease mode of inheritance is inaccurate. Under some conditions, problems result if one is not careful to consider what null hypothesis is being tested. We show that: (a) tests for which the null hypothesis assumes the absence of both linkage and association are independent of the true mode of inheritance; (b) likelihood ratio tests assuming either linkage or association under the null hypothesis may depend on the true mode of inheritance, leading to inconsistent parameter estimates, in particular under extremely deterministic models; (c) this problem cannot be eliminated by increasing sample size or adding population controls--as sample size increases, the chance of false positive inference goes to 100%; (d) this issue can lead to systematic false positive inference of association in regions of linkage. This is important because highly deterministic models are often used intentionally in model-based analyses because they can have more power than the true model, and are implicit in many model-free analysis methods.

A common approach to genetic mapping of loci for complex diseases is to perform a genome-wide association study (GWAS) by analyzing a vast number of SNP markers in cohorts of unrelated cases and controls. A direct motivation for the case-control design is that unrelated, affected individuals can be easier to collect than large families with multiple affected persons in the Western world. Despite its higher potential power, investigators have not actively pursued family ascertainment in part because of a dearth of methods for analyzing such correlated data on a large scale. We examine the statistical properties of several commonly used family-based association tests, as to their performance using real-life mixtures of families and singletons taken from our own migraine and schizophrenia studies, as well as population-based data for a complex trait simulated with the evolutionary phenogenetic simulator, ForSim. In virtually every situation, the full likelihood-based methods in the PSEUDOMARKER program outperformed those implemented in FBAT, GENEHUNTER TDT, PLINK (family-based options), HRR/HHRR, QTDT, TRANSMIT, UNPHASED, MENDEL, and LAMP. We further show that GWAS is much more powerful when family samples are used rather than unrelateds, on a genotype-by-genotype basis.

A decade ago, there was widespread enthusiasm for the prospects of genome-wide association studies to identify common variants related to common chronic diseases using samples of unrelated individuals from populations. Although technological advancements allow us to query more than a million SNPs across the genome at low cost, a disappointingly small fraction of the genetic portion of common disease etiology has been uncovered. This has led to the hypothesis that less frequent variants might be involved, stimulating a renaissance of the traditional approach of seeking genes using multiplex families from less diverse populations. However, by using the modern genotyping and sequencing technology, we can now look not just at linkage, but jointly at linkage and linkage disequilibrium (LD) in such samples. Software methods that can look simultaneously at linkage and LD in a powerful and robust manner have been lacking. Most algorithms cannot jointly analyze datasets involving families of varying structures in a statistically or computationally efficient manner. We have implemented previously proposed statistical algorithms in a user-friendly software package, PSEUDOMARKER. This paper is an announcement of this software package. We describe the motivation behind the approach, the statistical methods, and software, and we briefly demonstrate PSEUDOMARKER’s advantages over other packages by example.

Background The G protein-coupled receptor neuropeptide S receptor 1 (NPSR1) and its ligand neuropeptide S (NPS) form a signaling system mainly implicated in susceptibility to asthma and inflammatory disorders in humans and regulation of anxiety and arousal in rodents. We addressed here the role of NPS and NPSR1 as susceptibility genes for human anxiety disorders. Methods We performed comprehensive association analysis of genetic variants in NPS and NPSR1 in three independent study samples. We first studied a population-based sample (Health 2000, Finland) of 321 anxiety disorder patients and 1317 control subjects and subsequently a Spanish clinical panic disorder sample consisting of 188 cases and 315 control subjects. In addition, we examined a birth cohort of 2020 children (Barn Allergi Miljo Stockholm Epidemiologi [BAMSE], Sweden). We then tested whether alleles of the most significantly associated single nucleotide polymorphisms alter DNA-protein complex formation in electrophoretic mobility shift assays. Finally, we compared acute stress responses on the gene expression level in wild-type and Npsr1 −/− mice. Results We confirmed previously observed epidemiological association between anxiety and asthma in two population-based cohorts. Single nucleotide polymorphisms within NPS and NPSR1 associated with panic disorder diagnosis in the Finnish and Spanish samples and with parent-reported anxiety/depression in the BAMSE sample. Moreover, some of the implicated single nucleotide polymorphisms potentially affect transcription factor binding. Expression of neurotrophin-3, a neurotrophic factor connected to stress and panic reaction, was significantly downregulated in brain regions of stressed Npsr1 −/− mice, whereas interleukin-1 beta, an active stress-related immunotransmitter, was upregulated. Conclusions Our results suggest that NPS-NPSR1 signaling is likely involved in anxiety.

Our Lasker Prize winning article from 2000 was a discussion of the myriad factors that influence the success or failure of gene mapping studies, and in it we sug- gested that the reasons we geneticists have had so little success in unraveling the etiology of complex traits had more to do with biology than technology and that many of the decisions about future strategies were being made based on unrealis- tic and untested assumptions. At the time, the common variant/common disease (CVCD) hypothesis (Reich and Lander 2001) was widely believed to provide a

In the 20th century geneticists began to unravel some of the simpler aspects of the etiology of inherited diseases in humans. The theory of linkage analysis was developed and applied long before the advent of molecular biology, but only the technological advances of the second half of the 20th century made large-scale gene mapping with a dense genome-spanning set of markers a reality. More recently, the primary topic of interest has shifted from simple Mendelian diseases, for which genotypes of some gene are the cause of disease, to more complex diseases, for which genotypes of some set of genes together with environmental factors merely alter the probability that an individual gets the disease, although individual factors are typically insufficient to cause the disease outright. To this end, a great deal of dogma has evolved about the best way to skin this cat, although to date success has been minimal with any approach. We postulate that the main reason for this is a lack of attention to experimental design. Once the data have been ascertained, the most powerful statistical methods will not be able to salvage an inappropriately designed study (Andersen 1990). Each phenotype and/or population mandates its own individually tailored study design to maximize the chances of successful gene mapping. We suggest that careful consideration of the available data from real genotype-phenotype correlation studies (as opposed to oversimplified theoretically tractable models), and the practical feasibility of different ascertainment schemes dictate how one should proceed. In this review we review the theory and practice of gene mapping at the close of the 20th century, showing that most methods of linkage and linkage disequilibrium analysis are similar in a fundamental sense, with the differences being related more to study design and ascertainment than to technical details of the underlying statistical analysis. To this end, we propose a new focus in the field of statistical genetics that more explicitly highlights the primacy of study design as the means to increase power for gene mapping.

Here, we present the results of two genome-wide scans in two diverse populations in which a consistent use of recently introduced migraine-phenotyping methods detects and replicates a locus on 10q22-q23, with an additional independent replication. No genetic variants have been convincingly established in migraine, and although several loci have been reported, none of them has been consistently replicated. We employed the three known migraine-phenotyping methods (clinical end diagnosis, latent-class analysis, and trait-component analysis) with robust multiple testing correction in a large sample set of 1675 individuals from 210 migraine families from Finland and Australia. Genome-wide multipoint linkage analysis that used the Kong and Cox exponential model in Finns detected a locus on 10q22-q23 with highly significant evidence of linkage (LOD 7.68 at 103 cM in female-specific analysis). The Australian sample showed a LOD score of 3.50 at the same locus (100 cM), as did the independent Finnish replication study (LOD score 2.41, at 102 cM). In addition, four previously reported loci on 8q21, 14q21, 18q12, and Xp21 were also replicated. A shared-segment analysis of 10q22-q23 linked Finnish families identified a 1.6-9.5 cM segment, centered on 101 cM, which shows in-family homology in 95% of affected Finns. This region was further studied with 1323 SNPs. Although no significant association was observed, four regions warranting follow-up studies were identified. These results support the use of symptomology-based phenotyping in migraine and suggest that the 10q22-q23 locus probably contains one or more migraine susceptibility variants.

Background Human anxiety disorders are complex diseases with largely unknown etiology. We have taken a cross-species approach to identify genes that regulate anxiety-like behavior with inbred mouse strains that differ in their innate anxiety levels as a model. We previously identified 17 genes with expression levels that correlate with anxiety behavior across the studied strains. In the present study, we tested their 13 known human homologues as candidate genes for human anxiety disorders with a genetic association study. Methods We describe an anxiety disorder study sample derived from a Finnish population-based cohort and consisting of 321 patients and 653 carefully matched control subjects, all interviewed to obtain DSM-IV diagnoses. We genotyped altogether 208 single nucleotide polymorphisms (SNPs) (all non-synonymous SNPs, SNPs that alter potential microRNA binding sites, and gap-filling SNPs selected on the basis of HapMap information) from the investigated anxiety candidate genes. Results Specific alleles and haplotypes of six of the examined genes revealed some evidence for association ( p ≤ .01). The most significant evidence for association with different anxiety disorder subtypes were: p = .0009 with ALAD (δ-aminolevulinate dehydratase) in social phobia, p = .009 with DYNLL2 (dynein light chain 2) in generalized anxiety disorder, and p = .004 with PSAP (prosaposin) in panic disorder. Conclusions Our findings suggest that variants in these genes might predispose to specific human anxiety disorders. These results illustrate the potential utility of cross-species approaches in identification of candidate genes for psychiatric disorders.

A single-nucleotide variant, C/T(-13910), located 14 kb upstream of the lactase gene (LCT), has been shown to be completely correlated with lactase persistence (LP) in northern Europeans. Here, we analyzed the background of the alleles carrying the critical variant in 1,611 DNA samples from 37 populations. Our data show that the T(-13910) variant is found on two different, highly divergent haplotype backgrounds in the global populations. The first is the most common LP haplotype (LP H98) present in all populations analyzed, whereas the others (LP H8-H12), which originate from the same ancestral allelic haplotype, are found in geographically restricted populations living west of the Urals and north of the Caucasus. The global distribution pattern of LP T(-13910) H98 supports the Caucasian origin of this allele. Age estimates based on different mathematical models show that the common LP T(-13910) H98 allele (approximately 5,000-12,000 years old) is relatively older than the other geographically restricted LP alleles (approximately 1,400-3,000 years old). Our data about global allelic haplotypes of the lactose-tolerance variant imply that the T(-13910) allele has been independently introduced more than once and that there is a still-ongoing process of convergent evolution of the LP alleles in humans.

We have previously reported a robust association between an allelic haplotype of ‘Disrupted in Schizophrenia 1’ (DISC1) and schizophrenia in a nationwide collection of Finnish schizophrenia families. This specific DISC1 allele was later identified to associate with visual working memory, selectively in males. DISC1 association to schizophrenia has since been replicated in multiple independent study samples from different populations. In this study, we conditioned our sample of Finnish families for the presence of the Finnish tentative risk allele for DISC1 and re-analyzed our genome-wide scan data of 443 markers on the basis of this stratification. Two additional loci displayed an evidence of linkage (LOD >3) and included a locus on 16p13, proximal to the gene encoding NDE1, which has been shown to biologically interact with DISC1. Although none of the observed linkages remained significant after multiple test correction through simulation, further analysis of NDE1 revealed an association between a tag-haplotype and schizophrenia (P 5 0.00046) specific to females, which proved to be significant (P 5 0.011) after multiple test correction. Our finding would support the concept that initial gene findings in multifactorial diseases will assist in the identification of other components of complex genetic etiology. Notably, this and other converging lines of evidence underline the importance of DISC1-related functional pathways in the etiology of schizophrenia.

The International HapMap Project was proposed in order to quantify linkage disequilibrium (LD) relationships among human DNA polymorphisms in an assortment of populations, in order to facilitate the process of selecting a minimal set of markers that could capture most of the signal from the untyped markers in a genome-wide association study. The central dogma can be summarized by the argument that if a marker is in tight LD with a polymorphism that directly impacts disease risk, as measured by the metric r(2), then one would be able to detect an association between the marker and disease with sample size that was increased by a factor of 1/r(2) over that needed to detect the effect of the functional variant directly. This "fundamental theorem" holds, however, only if one assumes that the LD between loci and the etiological effect of the functional variant are independent of each other, that they are statistically independent of all other etiological factors (in exposure and action), that sampling is prospective, and that the estimates of r(2) are accurate. None of these are standard operating assumptions, however. We describe the ramifications of these implicit assumptions, and provide simple examples in which the effects of a functional variant could be unequivocally detected if it were directly genotyped, even as markers in high LD with the functional variant would never show association with disease, even in infinite sample sizes. Both theoretical and empirical refutation of the central dogma of genome-wide association studies is thus presented.

We previously reported a genomewide scan to identify autism-susceptibility loci in 110 multiplex families, showing suggestive evidence (P.01) for linkage to autism-spectrum disorders (ASD) on chromosomes 5, 8, 16, 19, and X and showing nominal evidence (P.05) on several additional chromosomes (2, 3, 4, 10, 11, 12, 15, 18, and 20). In this follow-up analysis we have increased the sample size threefold, while holding the study design constant, so that we now report 345 multiplex families, each with at least two siblings affected with autism or ASD phenotype. Along with 235 new multiplex families, 73 new microsatellite markers were also added in 10 regions, thereby increasing the marker density at these strategic locations from 10 cM to approximately 2 cM and bringing the total number of markers to 408 over the entire genome. Multipoint maximum LOD scores (MLS) obtained from affected-sib-pair analysis of all 345 families yielded suggestive evidence for linkage on chromosomes 17, 5, 11, 4, and 8 (listed in order by MLS) (P.01). The most significant findings were an MLS of 2.83 (P =.00029) on chromosome 17q, near the serotonin transporter (5-hydroxytryptamine transporter [5-HTT]), and an MLS of 2.54 (P =.00059) on 5p. The present follow-up genome scan, which used a consistent research design across studies and examined the largest ASD sample collection reported to date, gave either equivalent or marginally increased evidence for linkage at several chromosomal regions implicated in our previous scan but eliminated evidence for linkage at other regions.

Many promises have been made about the impact of the Human Genome Project on clinical practice and public health, yet despite massively funded efforts over the past decade, little headway has been made in elucidating the specific genetic factors which have major impact on the risk of developing common complex traits. There are two fundamental reasons for this abject failure as follows: 1) studies have been inadequately designed to identify such genetic risk factors; 2) the genetic factors that do exist are individually of small marginal importance, and are characterized by extensive heterogeneity. If 2) is the truth, there is little we can do about it, so we emphasize the importance of 1) in this article, while recognizing that 2) probably is not far from the truth. Genetic studies, in contrast to epidemiological studies, use confounding and ascertainment bias to help identify weak etiologic signal due to genes, since gene mapping is fundamentally a hypothesis-free science. This strategy makes it possible to identify genetic risk factors, but makes it impossible to quantify the size of their effect on risk. Classical epidemiological study designs are of minimal value for gene identification, but may be of use in estimation of the effect size of genetic risk factors once they are identified in more appropriately designed genetic studies. However, if the effects are so weak that we need this strong, systematic ascertainment bias to find them, their relevance to public health may be of questionable immediate value, raising many questions about the rhetoric and promises being made to the public as justification for 'big science' approaches to dissecting the hypothetical role of genes in complex traits.

Adult-type hypolactasia, also known as lactase non-persistence (lactose intolerance), is a common autosomal recessive condition resulting from the physiological decline in activity of the lactase-phlorizin hydrolase (LPH) in intestinal cells after weaning. LPH hydrolyzes lactose into glucose and galactose. Sequence analyses of the coding and promoter regions of LCT, the gene encoding LPH, has revealed no DNA variations correlating with lactase non-persistence. An associated haplotype spanning LCT, as well as a distinct difference in the transcript levels of 'non-persistence' and 'persistence' alleles in heterozygotes, suggest that a cis-acting element contributes to the lactase non-persistence phenotype. Using linkage disequilibrium (LD) and haplotype analysis of nine extended Finnish families, we restricted the locus to a 47-kb interval on 2q21. Sequence analysis of the complete region and subsequent association analyses revealed that a DNA variant, C/T-13910, roughly 14 kb upstream from the LCT locus, completely associates with biochemically verified lactase non-persistence in Finnish families and a sample set of 236 individuals from four different populations. A second variant, G/A-22018, 8 kb telomeric to C/T-13910, is also associated with the trait in 229 of 236 cases. Prevalence of the C/T-13910 variant in 1,047 DNA samples is consistent with the reported prevalence of adult-type hypolactasia in four different populations. That the variant (C/T-13910) occurs in distantly related populations indicates that it is very old.

In inflammatory bowel diseases (IBD), certain chromosomal candidate loci have been repeatedly identified by independent studies in different populations. To investigate the contribution of the loci on chromosomes 1, 3, 7, 12, 14, and 16 to the susceptibility of IBD in Finnish population, where the predominant feature is the excess of ulcerative colitis (UC) families compared to Crohn’s disease (CD) families, we carried out linkage analyses using 93 Finnish, multiply-affected IBD families. We observed nominal evidence for linkage to chromosome 3p21, consistent with earlier reports. The lod scores peaked at D3S2432, with a maximum two-point lod score of 1.68 (P=0.0027). In addition, we studied whether risk of IBD is associated with functional variants of two positional candidate genes; the chemokine receptor CCR5 gene on chromosome 3p21 and the interleukin-4 receptor a-subunit gene (IL4RA) on chromosome 16. We did not find any significant correlation between a 32bp deletion variant of CCR5 or a single nucleotide change A1902G (Gln576Arg) of IL4RA, and IBD phenotypes, with the exception that in the UC group homozygosity for the G1902 allele of IL4RA was less frequent (0.019 vs 0.049, P=0.038). In conclusion, our study, carried out in a genetically homogenous population, suggests that chromosome 3 may contain a susceptibility gene for IBD. European Journal of Human Genetics (2001) 9, 328 ‐ 334.

Epidemiological studies have shown that genetic factors contribute to the etiology of the common and serious pregnancy-specific disorder pre-eclampsia (PE)/eclampsia (E). Candidate-gene studies have provided evidence (albeit controversial) of linkage to several genes, including angiotensinogen on 1q42-43 and eNOS on 7q36. A recent medium-density genome scan in Icelandic families identified significant linkage to D2S286 (at 94.05 cM) on chromosome 2p12 and suggestive linkage to D2S321 (at 157.5 cM) on chromosome 2q23. In the present article, the authors report the results of a medium-density genome scan in 34 families, representing 121 affected women, from Australia and New Zealand. Multipoint nonparametric linkage analysis, using the GENEHUNTER-PLUS program, showed suggestive evidence of linkage to chromosome 2 (LOD=2.58), at 144.7 cM, between D2S112 and D2S151, and to chromosome 11q23-24, between D11S925 and D11S4151 (LOD=2.02 at 121.3 cM). Given the limited precision of estimates of the map location of disease-predisposing loci for complex traits, the present finding on chromosome 2 is consistent with the finding from the Icelandic study, and it may represent evidence of the same locus segregating in the population from Australia and New Zealand. The authors propose that the PE/E-linked locus on chromosome 2p should be designated the “PREG1” (pre-eclampsia, eclampsia gene 1) locus.

Linkage disequilibrium (LD), non-random association of alleles at closely linked chromosomal loci, has been used as a tool in the identification of disease alleles, and this has led to an improved understanding of pathology in many monogenic Mendelian human diseases. We are currently moving from the mapping and identification of monogenic disease loci to attempts at identifying loci involved in predisposition to multifactorial diseases. In the selection of ascertainment strategies in the studies of these complex diseases, the extent of background LD in different populations is an important consideration. Here, we compare the extent of LD among the alleles of linked loci in a randomly ascertained sample of individuals from the Finnish population and a set of individuals ascertained from the region of Kuusamo, a small sub-population, founded some 13 generations ago, which has experienced very little subsequent immigration. Thirty-three microsatellite loci were genotyped in chromosomal regions on 13q, 19q, 21q, Xq, and Xp. The genetic diversity of these loci was determined separately in the general Finnish sample and in the Kuusamo sample. The X-chromosomal loci are characterised by higher levels of LD in the samples from Kuusamo than in the much larger (and older) general population of Finland, whereas in alleles of autosomal loci very little LD was seen in either of these two samples. European Journal of Human Genetics (2000) 8, 604‐612.

The admixture test of linkage heterogeneity is the most often and most successfully applied oligogenic-model linkage and/or LD analysis method. Full two-locus model linkage analysis is possible, but can be computationally intensive and difficult to interpret because of the need to specify so many indeterminate parameters. A novel, computationally efficient method is proposed for combining single locus lod scores which can allow for varying degrees of epistatic interaction. This method can be applied to two-point or multipoint (using complex-valued recombination fractions) linkage and/or linkage disequilibrium analysis to jointly test for multiple unlinked disease loci. Unlike the traditional admixture test, this algorithm permits joint analysis of multiple disease loci with different modes of inheritance for each, and can be applied to 'model-free' analysis as well through the use of 'pseudomarkers'. Software is available for computation of the various likelihood ratio tests described, for comparison of a variety of possible hypotheses regarding locus homogeneity, locus heterogeneity, and epistasis.

In linkage and linkage disequilibrium (LD) analysis of complex multifactorial phenotypes, various types of errors can greatly reduce the chance of successful gene localization. The power of such studies—even in the absence of errors—is quite low, and, accordingly, their robustness to errors can be poor, especially in multipoint analysis. For this reason, it is important to deal with the ramifications of errors up front, as part of the analytical strategy. In this study, errors in the characterization of marker-locus parameters—including allele frequencies, haplotype frequencies (i.e., LD between marker loci), recombination fractions, and locus order—are dealt with through the use of profile likelihoods maximized over such nuisance parameters. It is shown that the common practice of assuming fixed, erroneous values for such parameters can reduce the power and/or increase the probability of obtaining false positive results in a study. The effects of errors in assumed parameter values are generally more severe when a larger number of less informative marker loci, like the highly-touted single nucleotide polymorphisms (SNPs), are analyzed jointly than when fewer but more informative marker loci, such as microsatellites, are used. Rather than fixing inaccurate values for these parameters a priori, we propose to treat them as nuisance parameters through the use of profile likelihoods. It is demonstrated that the power of linkage and/or LD analysis can be increased through application of this technique in situations where parameter values cannot be specified with a high degree of certainty.

There is a lot of confusion in the literature about the “differences” between “model-based” and “model-free” methods and about which approach is better suited for detection of the genes predisposing to complex multifactorial phenotypes. By starting from first principles, we demonstrate that the differences between the two approaches have more to do with study design than statistical analysis. When simple data structures are repeatedly ascertained, no assumptions about the genotype-phenotype relationship need to be made for the analysis to be powerful, since simple data structures admit only a small number of df. When more complicated and/or heterogeneous data structures are ascertained, however, the number of df in the underlying probability model is too large to have a powerful, truly “model-free” test. So-called “model-free” methods typically simplify the underlying probability model by implicitly assuming that, in some sense, all meioses connecting two affected individuals are informative for linkage with identical probability and that the affected individuals in a pedigree share as many disease-predisposing alleles as possible. By contrast, “model-based” methods add structure to the underlying parameter space by making assumptions about the genotype-phenotype relationship, making it possible to probabilistically assign disease-locus genotypes to all individuals in the data set on the basis of the observed phenotypes. In this study, we demonstrate the equivalence of these two approaches in a variety of situations and exploit this equivalence to develop more powerful and efficient likelihood-based analogues of “model-free” tests of linkage and/or linkage disequilibrium. Through the use of a “pseudomarker” locus to structure the space of observations, sib-pairs, triads, and singletons can be analyzed jointly, which will lead to tests that are more well-behaved, efficient, and powerful than traditional “model-free” tests such as the affected sib-pair, transmission/disequilibrium, haplotype relative risk, and case-control tests. Also described is an extension of this approach to large pedigrees, which, in practice, is equivalent to affected relative-pair analysis. The proposed methods are equally applicable to two-point and multipoint analysis (using complex-valued recombination fractions).