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Objectives: We aimed to determine whether the common variation at the chromogranin A (CHGA) locus increases susceptibility to hypertension.Background: CHGA regulates catecholamine storage and release. Previously we systematically identified genetic variants across CHGA.Methods: We carried out dense genotyping across the CHGA locus in >1,000 individuals with the most extreme blood pressures (BPs) in the population, as well as twin pairs with autonomic phenotypes. We also characterized the function of a trait-associated 3'-untranslated region (3'-UTR) variant with transfected CHGA 3'-UTR/luciferase reporter plasmids.Results: CHGA was overexpressed in patients with hypertension, especially hypertensive men, and CHGA predicted catecholamines. In individuals with extreme BPs, CHGA genetic variants predicted BP, especially in men, with a peak association occurring in the 3'-UTR at C+87T, accounting for up to approximately 12/ approximately 9 mm Hg. The C+87T genotype predicted CHGA secretion in vivo, with the +87T allele (associated with lower BP) also diminishing plasma CHGA by approximately 10%. The C+87T 3'-UTR variant also predicted the BP response to environmental (cold) stress; the same allele (+87T) that diminished basal BP in the population also decreased the systolic BP response to stress by approximately 12 mm Hg, and the response was smaller in women (by approximately 6 mm Hg). In a chromaffin cell-transfected CHGA 3'-UTR/luciferase reporter plasmid, the +87T allele associated with lower BP also decreased reporter expression by approximately 30%. In cultured chromaffin cells, reducing endogenous CHGA expression by small interfering ribonucleic acid caused approximately two-thirds depletion of catecholamine storage vesicles.Conclusions: Common variant C+87T in the CHGA 3'-UTR is a functional polymorphism causally associated with hypertension especially in men of the population, and we propose steps ("intermediate phenotypes") whereby in a sex-dependent fashion this genetic variant influences the ultimate disease trait. These observations suggest new molecular strategies to probe the pathophysiology, risk, and rational treatment of hypertension. [ABSTRACT FROM AUTHOR]

Objectives: We asked whether naturally occurring genetic variation at the human NPY1R locus alters autonomic traits that might predispose individuals to cardiovascular disease.Background: Neuropeptide Y (NPY) interacts with the Y(1) receptor, NPY1R, to control adrenergic activity and blood pressure (BP).Methods: We searched for polymorphism at NPY1R by systematic resequencing in ethnically diverse people. There were 376 twins/siblings who were evaluated for heritable autonomic traits: baroreflex function and pressor response to environmental stress.Results: The common NPY1R variant A+1050G in the 3'-untranslated region (3'-UTR) predicted baroreceptor slope (p = 0.014-0.047) and BP change to cold stress (p = 0.0091-0.016), with minor allele homozygotes displaying blunted slope and exaggerated pressor response. In 936 individuals with the most extreme BPs in the population, not only 3'-UTR A+1050G (p = 1.2 x 10(-4)) but also promoter A-585T (p = 0.001) affected both systolic BP and diastolic BP, in interactive fashion (p = 0.007), with combined homozygotes showing the highest diastolic BP (>20 mm Hg). The 3'-UTR variant +1050G decreased expression of a transfected luciferase reporter/NPY1R 3'-UTR plasmid; promoter variant A-585 also decreased expression of an NPY1R promoter/luciferase reporter. Thus, alleles that increased BP in vivo (3'-UTR +1050G, promoter A-585) also decreased NPY1R expression in cella. Computational alignment showed that A+1050G disrupted a microRNA motif.Conclusions: Our results indicate that naturally occurring genetic variation at the NPY1R locus has implications for heritable autonomic control of the circulation, and ultimately, for systemic hypertension. The findings suggest novel pathophysiological links between the NPY1R locus, autonomic activity, and blood pressure, and suggest new strategies to approach the mechanism, diagnosis, and treatment of systemic hypertension. [ABSTRACT FROM AUTHOR]

Growth differentiation factor 15 (GDF15) is a secreted protein that regulates food intake, body weight and stress responses in pre-clinical models 1 . The physiological function of GDF15 in humans remains unclear. Pharmacologically, GDF15 agonism in humans causes nausea without accompanying weight loss 2 , and GDF15 antagonism is being tested in clinical trials to treat cachexia and anorexia. Human genetics point to a role for GDF15 in hyperemesis gravidarum, but the safety or impact of complete GDF15 loss, particularly during pregnancy, is unknown 3-7 . Here we show the absence of an overt phenotype in human GDF15 loss-of-function carriers, including stop gains, frameshifts and the fully inactivating missense variant C211G 3 . These individuals were identified from 75,018 whole-exome/genome-sequenced participants in the Pakistan Genomic Resource 8,9 and recall-by-genotype studies with family-based recruitment of variant carrier probands. We describe 8 homozygous ('knockouts') and 227 heterozygous carriers of loss-of-function alleles, including C211G. GDF15 knockouts range in age from 31 to 75 years, are fertile, have multiple children and show no consistent overt phenotypes, including metabolic dysfunction. Our data support the hypothesis that GDF15 is not required for fertility, healthy pregnancy, foetal development or survival into adulthood. These observations support the safety of therapeutics that block GDF15., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The genetic factors of stroke in South Asians are largely unexplored. Exome-wide sequencing and association analysis (ExWAS) in 75 K Pakistanis identified NM_000435.3(NOTCH3):c.3691 C > T, encoding the missense amino acid substitution p.Arg1231Cys, enriched in South Asians (alternate allele frequency = 0.58% compared to 0.019% in Western Europeans), and associated with subcortical hemorrhagic stroke [odds ratio (OR) = 3.39, 95% confidence interval (CI) = [2.26, 5.10], p = 3.87 × 10 -9 ), and all strokes (OR [CI] = 2.30 [1.77, 3.01], p = 7.79 × 10 -10 ). NOTCH3 p.Arg231Cys was strongly associated with white matter hyperintensity on MRI in United Kingdom Biobank (UKB) participants (effect [95% CI] in SD units = 1.1 [0.61, 1.5], p = 3.0 × 10 -6 ). The variant is attributable for approximately 2.0% of hemorrhagic strokes and 1.1% of all strokes in South Asians. These findings highlight the value of diversity in genetic studies and have major implications for genomic medicine and therapeutic development in South Asian populations., (© 2024. The Author(s).)

Autoimmune conditions have been reported among patients with cysteine-altering NOTCH3 variants and CADASIL. This study aimed to investigate the occurrence of autoimmune illnesses and markers of inflammation in such populations. Cases were identified who had a NOTCH3 cysteine-altering variant from the Geisinger MyCode ® Community Health Initiative (MyCode ® ). We further performed external validation using the UK Biobank cohort. A cohort of 121 individuals with a NOTCH3 cysteine-altering variant from MyCode ® was compared to a control group with no non-synonymous variation in NOTCH3 ( n = 184). Medical records were evaluated for inflammatory markers and autoimmune conditions, which were grouped by the organ systems involved. A similar analysis was conducted using data from the UK Biobank ( n ~450,000). An overall increase in inflammatory markers among participants with a NOTCH3 cysteine-altering variant was observed when compared to an age- and sex-matched MyCode ® control group (out of participants with laboratory testing: 50.9% versus 26.7%; p = 0.0047; out of total participants: 23.1% versus 10.9%; p = 0.004). Analysis of UK Biobank data indicated any autoimmune diagnosis (1.63 [1.14, 2.09], p = 2.665 × 10 -3 ) and multiple sclerosis (3.42 [1.67, 6.02], p = 9.681 × 10 -4 ) are associated with a NOTCH3 cysteine-altering variant in any domain. Our findings suggest a possible association between NOTCH3 cysteine-altering variants and autoimmune conditions.

Risk genes for Mendelian (single-gene) disorders (SGDs) are consistent across populations, but pathogenic risk variants that cause SGDs are typically population-private. The goal was to develop "QChip1," an inexpensive genotyping microarray to comprehensively screen newborns, couples, and patients for SGD risk variants in Qatar, a small nation on the Arabian Peninsula with a high degree of consanguinity. Over 10 8 variants in 8445 Qatari were identified for inclusion in a genotyping array containing 165,695 probes for 83,542 known and potentially pathogenic variants in 3438 SGDs. QChip1 had a concordance with whole-genome sequencing of 99.1%. Testing of QChip1 with 2707 Qatari genomes identified 32,674 risk variants, an average of 134 pathogenic alleles per Qatari genome. The most common pathogenic variants were those causing homocystinuria (1.12% risk allele frequency), and Stargardt disease (2.07%). The majority (85%) of Qatari SGD pathogenic variants were not present in Western populations such as European American, South Asian American, and African American in New York City and European and Afro-Caribbean in Puerto Rico; and only 50% were observed in a broad collection of data across the Greater Middle East including Kuwait, Iran, and United Arab Emirates. This study demonstrates the feasibility of developing accurate screening tools to identify SGD risk variants in understudied populations, and the need for ancestry-specific SGD screening tools., (© 2022. The Author(s).)

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by detectable hematopoietic-associated gene mutations in a person without evidence of hematologic malignancy. We sought to identify additional cancer-presenting mutations useable for CHIP detection by performing a data mining analysis of 48 somatic mutation studies reporting mutations at diagnoses of 7,430 adult and pediatric patients with hematologic malignancies. Following extraction of 20,141 protein-altering mutations, we identified 434 significantly recurrent mutation hotspots, 364 of which occurred at loci confidently assessable for CHIP. We then performed an additional large-scale analysis of whole exome sequencing data from 4,538 persons belonging to three non-cancer cohorts for clonal mutations. We found the combined cohort prevalence of CHIP with mutations identical to those reported at blood cancer mutation hotspots to be 1.8%, and that some of these CHIP mutations occurred in children. Our findings may help to improve CHIP detection and pre-cancer surveillance for both children and adults., Competing Interests: The authors declare no conflict of interests.

Obesity, a major risk factor for metabolic disorders, is highly prevalent in Qatari population. Maternal transmission of obesity traits can be significant; for example, X haplogroup is known to be associated with lower BMI and body fat mass in Northern Europeans and T haplogroup which is a sister haplogroup of J is known to be associated with obesity in Caucasian subjects from Austria and Southern Italy. We aimed to delineate the mitochondrial haplogroups and variants associated with obesity in Qatari population. Mitochondrial genomes of 864 Qatari individuals were extracted from whole exome sequencing data with an average coverage of 77X. We distributed the participants into 2 sub-cohorts: obese (BMI ≥ 30) and non-obese (BMI < 30); the mean value of BMI from these two groups were 36.5 ± 5.7 and 26.5 ± 2.6, respectively. Mitochondrial haplogroup profiling followed by uni- and multivariant association tests adjusted for covariates were performed. Qatari individuals with mitochondrial haplogroup J had an increased (twofold) risk of obesity (odds ratio [OR] 1.925; 95% CI 1.234-3.002; P = 0.0038; the Bonferroni adjusted P value threshold is 0.0041), whereas the individuals with haplogroup X were at low risk of obesity (OR 0.387; 95% CI 0.175-0.857; P = 0.019). Further, a set of 38 mitochondrial variants were found to be associated (at P ≤ 0.05) with obesity in models adjusted for age, sex and haplogroup.

Background: The main goal of this collaborative effort is to provide genome-wide data for the previously underrepresented population in Eastern Europe, and to provide cross-validation of the data from genome sequences and genotypes of the same individuals acquired by different technologies. We collected 97 genome-grade DNA samples from consented individuals representing major regions of Ukraine that were consented for public data release. BGISEQ-500 sequence data and genotypes by an Illumina GWAS chip were cross-validated on multiple samples and additionally referenced to 1 sample that has been resequenced by Illumina NovaSeq6000 S4 at high coverage., Results: The genome data have been searched for genomic variation represented in this population, and a number of variants have been reported: large structural variants, indels, copy number variations, single-nucletide polymorphisms, and microsatellites. To our knowledge, this study provides the largest to-date survey of genetic variation in Ukraine, creating a public reference resource aiming to provide data for medical research in a large understudied population., Conclusions: Our results indicate that the genetic diversity of the Ukrainian population is uniquely shaped by evolutionary and demographic forces and cannot be ignored in future genetic and biomedical studies. These data will contribute a wealth of new information bringing forth a wealth of novel, endemic and medically related alleles., (© The Author(s) 2021. Published by Oxford University Press GigaScience.)

Islands have been used as model systems for studies of speciation and extinction since Darwin published his observations about finches found on the Galapagos. Amazon parrots inhabiting the Greater Antillean Islands represent a fascinating model of species diversification. Unfortunately, many of these birds are threatened as a result of human activity and some, like the Puerto Rican parrot, are now critically endangered. In this study we used a combination of de novo and reference-assisted assembly methods, integrating it with information obtained from related genomes to perform genome reconstruction of three amazon species. First, we used whole genome sequencing data to generate a new de novo genome assembly for the Puerto Rican parrot ( Amazona vittata ). We then improved the obtained assembly using transcriptome data from Amazona ventralis and used the resulting sequences as a reference to assemble the genomes Hispaniolan ( A. ventralis ) and Cuban ( Amazona leucocephala ) parrots. Finally, we, annotated genes and repetitive elements, estimated genome sizes and current levels of heterozygosity, built models of demographic history and provided interpretation of our findings in the context of parrot evolution in the Caribbean.

Purpose: Nonobstructive azoospermia (NOA) affects 1% of the male population; however, despite state-of-the-art clinical assessment, for most patients the cause is unknown. We capitalized on an analysis of multiplex families in the Middle East to identify highly penetrant genetic causes., Methods: We used whole-exome sequencing (WES) in 8 consanguineous families and combined newly discovered genes with previously reported ones to create a NOA gene panel, which was used to identify additional variants in 75 unrelated idiopathic NOA subjects and 74 fertile controls., Results: In five of eight families, we identified rare deleterious recessive variants in CCDC155, NANOS2, SPO11, TEX14, and WNK3 segregating with disease. These genes, which are novel to human NOA, have remarkable testis-specific expression, and murine functional evidence supports roles for them in spermatogenesis. Among 75 unrelated NOA subjects, we identified 4 (~5.3%) with additional recessive variants in these newly discovered genes and 6 with deleterious variants in previously reported NOA genes, yielding an overall genetic etiology for 13.3% subjects versus 0 fertile controls (p = 0.001)., Conclusion: NOA affects millions of men, many of whom remain idiopathic despite extensive laboratory evaluation. The genetic etiology for a substantial fraction of these patients (>50% familial and >10% sporadic) may be discovered by WES at the point of care.

Background: Type 2 diabetes (T2D) susceptibility is influenced by genetic and lifestyle factors. To date, the majority of genetic studies of T2D have been in populations of European and Asian descent. The focus of this study is on genetic variations underlying T2D in Qataris, a population with one of the highest incidences of T2D worldwide., Results: Illumina HiSeq exome sequencing was performed on 864 Qatari subjects (574 T2D cases, 290 controls). Sequence kernel association test (SKAT) gene-based analysis identified an association for low frequency potentially deleterious variants in 6 genes. However, these findings were not replicated by SKAT analysis in an independent cohort of 12,699 exomes, primarly due to the absence of low frequency potentially deleterious variants in 5 of the 6 genes. Interestingly one of the genes identified, catenin beta 1 (CTNNB1, β-catenin), is the key effector of the Wnt pathway and interacts with the nuclear receptor transcription factor 7-like 2 (TCF7L2), variants which are the most strongly associated with risk of developing T2D worldwide. Single variant analysis did not identify any associated variants, suggesting the SKAT association signal was not driven by individual variants. None of the 6 associated genes were among 634 previously described T2D genes., Conclusions: The observation that genes not previously linked to T2D in prior studies of European and Asian populations are associated with T2D in Qatar provides new insights into the complexity of T2D pathogenesis and emphasizes the importance of understudied populations when assessing genetic variation in the pathogenesis of common disorders., Competing Interests: The authors have declared that no competing interests exist.

Metabolomics-genome-wide association studies (mGWAS) have uncovered many metabolic quantitative trait loci (mQTLs) influencing human metabolic individuality, though predominantly in European cohorts. By combining whole-exome sequencing with a high-resolution metabolomics profiling for a highly consanguineous Middle Eastern population, we discover 21 common variant and 12 functional rare variant mQTLs, of which 45% are novel altogether. We fine-map 10 common variant mQTLs to new metabolite ratio associations, and 11 common variant mQTLs to putative protein-altering variants. This is the first work to report common and rare variant mQTLs linked to diseases and/or pharmacological targets in a consanguineous Arab cohort, with wide implications for precision medicine in the Middle East.

Obsessive compulsive disorder (OCD) is substantially heritable, but few molecular genetic risk factors have been identified. Knockout mice lacking SLIT and NTRK-Like Family, Member 5 (SLITRK5) display OCD-like phenotypes including serotonin reuptake inhibitor-sensitive pathologic grooming, and corticostriatal dysfunction. Thus, mutations that impair SLITRK5 function may contribute to the genetic risk for OCD. We re-sequenced the protein-coding sequence of the human SLITRK5 gene (SLITRK5) in three hundred and seventy seven OCD subjects and compared rare non-synonymous mutations (RNMs) in that sample with similar mutations in the 1000 Genomes database. We also performed in silico assessments and in vitro functional synaptogenesis assays on the Slitrk5 mutations identified. We identified four RNM's among these OCD subjects. There were no significant differences in the prevalence or in silico effects of rare non-synonymous mutations in the OCD sample versus controls. Direct functional testing of recombinant SLITRK5 proteins found that all mutations identified in OCD subjects impaired synaptogenic activity whereas none of the pseudo-matched mutations identified in 1000 Genomes controls had significant effects on SLITRK5 function (Fisher's exact test P = 0.028). These results demonstrate that rare functional mutations in SLITRK5 contribute to the genetic risk for OCD in human populations. They also highlight the importance of biological characterization of allelic effects in understanding genotype-phenotype relationships as there were no statistical differences in overall prevalence or bioinformatically predicted effects of OCD case versus control mutations. Finally, these results converge with others to highlight the role of aberrant synaptic function in corticostriatal neurons in the pathophysiology of OCD., Competing Interests: The authors have declared that no competing interests exist.

Background: The prevalence of type 2 diabetes (T2D) is increasing in the Middle East. However, the genetic risk factors for T2D in the Middle Eastern populations are not known, as the majority of studies of genetic risk for T2D are in Europeans and Asians., Methods: All subjects were ≥3 generation Qataris. Cases with T2D (n = 1,124) and controls (n = 590) were randomly recruited and assigned to the 3 known Qatari genetic subpopulations [Bedouin (Q1), Persian/South Asian (Q2) and African (Q3)]. Subjects underwent genotyping for 37 single nucleotide polymorphisms (SNPs) in 29 genes known to be associated with T2D in Europeans and/or Asian populations, and an additional 27 tag SNPs related to these susceptibility loci. Pre-study power analysis suggested that with the known incidence of T2D in adult Qataris (22%), the study population size would be sufficient to detect significant differences if the SNPs were risk factors among Qataris, assuming that the odds ratio (OR) for T2D SNPs in Qatari's is greater than or equal to the SNP with highest known OR in other populations., Results: Haplotype analysis demonstrated that Qatari haplotypes in the region of known T2D risk alleles in Q1 and Q2 genetic subpopulations were similar to European haplotypes. After Benjamini-Hochberg adjustment for multiple testing, only two SNPs (rs7903146 and rs4506565), both associated with transcription factor 7-like 2 (TCF7L2), achieved statistical significance in the whole study population. When T2D subjects and control subjects were assigned to the known 3 Qatari subpopulations, and analyzed individually and with the Q1 and Q2 genetic subpopulations combined, one of these SNPs (rs4506565) was also significant in the admixed group. No other SNPs associated with T2D in all Qataris or individual genetic subpopulations., Conclusions: With the caveats of the power analysis, the European/Asian T2D SNPs do not contribute significantly to the high prevalence of T2D in the Qatari population, suggesting that the genetic risks for T2D are likely different in Qataris compared to Europeans and Asians.

Reaching the full potential of precision medicine depends on the quality of personalized genome interpretation. In order to facilitate precision medicine in regions of the Middle East and North Africa (MENA), a population-specific genome for the indigenous Arab population of Qatar (QTRG) was constructed by incorporating allele frequency data from sequencing of 1,161 Qataris, representing 0.4% of the population. A total of 20.9 million single nucleotide polymorphisms (SNPs) and 3.1 million indels were observed in Qatar, including an average of 1.79% novel variants per individual genome. Replacement of the GRCh37 standard reference with QTRG in a best practices genome analysis workflow resulted in an average of 7* deeper coverage depth (an improvement of 23%) and 756,671 fewer variants on average, a reduction of 16% that is attributed to common Qatari alleles being present in QTRG. The benefit for using QTRG varies across ancestries, a factor that should be taken into consideration when selecting an appropriate reference for analysis.

We report the sequences of 1,244 human Y chromosomes randomly ascertained from 26 worldwide populations by the 1000 Genomes Project. We discovered more than 65,000 variants, including single-nucleotide variants, multiple-nucleotide variants, insertions and deletions, short tandem repeats, and copy number variants. Of these, copy number variants contribute the greatest predicted functional impact. We constructed a calibrated phylogenetic tree on the basis of binary single-nucleotide variants and projected the more complex variants onto it, estimating the number of mutations for each class. Our phylogeny shows bursts of extreme expansion in male numbers that have occurred independently among each of the five continental superpopulations examined, at times of known migrations and technological innovations.

An open question in the history of human migration is the identity of the earliest Eurasian populations that have left contemporary descendants. The Arabian Peninsula was the initial site of the out-of-Africa migrations that occurred between 125,000 and 60,000 yr ago, leading to the hypothesis that the first Eurasian populations were established on the Peninsula and that contemporary indigenous Arabs are direct descendants of these ancient peoples. To assess this hypothesis, we sequenced the entire genomes of 104 unrelated natives of the Arabian Peninsula at high coverage, including 56 of indigenous Arab ancestry. The indigenous Arab genomes defined a cluster distinct from other ancestral groups, and these genomes showed clear hallmarks of an ancient out-of-Africa bottleneck. Similar to other Middle Eastern populations, the indigenous Arabs had higher levels of Neanderthal admixture compared to Africans but had lower levels than Europeans and Asians. These levels of Neanderthal admixture are consistent with an early divergence of Arab ancestors after the out-of-Africa bottleneck but before the major Neanderthal admixture events in Europe and other regions of Eurasia. When compared to worldwide populations sampled in the 1000 Genomes Project, although the indigenous Arabs had a signal of admixture with Europeans, they clustered in a basal, outgroup position to all 1000 Genomes non-Africans when considering pairwise similarity across the entire genome. These results place indigenous Arabs as the most distant relatives of all other contemporary non-Africans and identify these people as direct descendants of the first Eurasian populations established by the out-of-Africa migrations., (© 2016 Rodriguez-Flores et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: The populations of the Arabian Peninsula remain the least represented in public genetic databases, both in terms of single nucleotide variants and of larger genomic mutations. We present the first high-resolution copy number variation (CNV) map for a Gulf Arab population, using a hybrid approach that integrates array genotyping intensity data and next-generation sequencing reads to call CNVs in the Qatari population., Methods: CNVs were detected in 97 unrelated Qatari individuals by running two calling algorithms on each of two primary datasets: high-resolution genotyping (Illumina Omni 2.5M) and high depth whole-genome sequencing (Illumina PE 100bp). The four call-sets were integrated to identify high confidence CNV regions, which were subsequently annotated for putative functional effect and compared to public databases of CNVs in other populations. The availability of genome sequence was leveraged to identify tagging SNPs in high LD with common deletions in this population, enabling their imputation from genotyping experiments in the future., Results: Genotyping intensities and genome sequencing data from 97 Qataris were analyzed with four different algorithms and integrated to discover 16,660 high confidence CNV regions (CNVRs) in the total population, affecting ~28 Mb in the median Qatari genome. Up to 40% of all CNVs affected genes, including novel CNVs affecting Mendelian disease genes, segregating at different frequencies in the 3 major Qatari subpopulations, including those with Bedouin, Persian/South Asian, and African ancestry. Consistent with high consanguinity levels in the Bedouin subpopulation, we found an increased burden for homozygous deletions in this group. In comparison to known CNVs in the comprehensive Database of Genomic Variants, we found that 5% of all CNVRs in Qataris were completely novel, with an enrichment of CNVs affecting several known chromosomal disorder loci and genes known to regulate sugar metabolism and type 2 diabetes in the Qatari cohort. Finally, we leveraged the availability of genome sequence to find suitable tagging SNPs for common deletions in this population., Conclusion: We combine four independently generated datasets from 97 individuals to study CNVs for the first time at high-resolution in a Gulf Arab population.

Apolipoprotein E, a protein component of blood lipid particles, plays an important role in lipid transport. Different mutations in the apolipoprotein E gene have been associated with various clinical phenotypes. In an initiated study of Qataris, we observed that 17% of the African-derived genetic subgroup were heterozygotes for a rare Arg145Cys (R145C) variant that functions as a dominant trait with incomplete penetrance associated with type III hyperlipoproteinemia. On the basis of this observation, we hypothesized that the R145C polymorphism might be common in African-derived populations. The prevalence of the R145C variant was assessed worldwide in the "1000 Genomes Project" and in 1,012 whites and 1,226 African-Americans in New York, New York. The 1000 Genomes Project data demonstrated that the R145C polymorphism is rare in non-African-derived populations but present in 5% to 12% of Sub-Saharan African-derived populations. The R145C polymorphism was also rare in New York whites (1 of 1,012, 0.1%); however, strikingly, 53 of the 1,226 New York African-Americans (4.3%) were R145C heterozygotes. The lipid profiles of the Qatari and New York R145C heterozygotes were compared with those of controls. The Qatari R145C subjects had higher triglyceride levels than the Qatari controls (p <0.007) and the New York African-American R145C subjects had an average of 52% greater fasting triglyceride levels than the New York African-American controls (p <0.002). From these observations, likely millions of people worldwide derived from Sub-Saharan Africans are apolipoprotein E R145C. In conclusion, although larger epidemiologic studies are necessary to determine the long-term consequences of this polymorphism, the available evidence suggests it is a common cause of a mild triglyceride dyslipidemia., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Exome sequencing of families of related individuals has been highly successful in identifying genetic polymorphisms responsible for Mendelian disorders. Here, we demonstrate the value of the reverse approach, where we use exome sequencing of a sample of unrelated individuals to analyze allele frequencies of known causal mutations for Mendelian diseases. We sequenced the exomes of 100 individuals representing the three major genetic subgroups of the Qatari population (Q1 Bedouin, Q2 Persian-South Asian, Q3 African) and identified 37 variants in 33 genes with effects on 36 clinically significant Mendelian diseases. These include variants not present in 1000 Genomes and variants at high frequency when compared with 1000 Genomes populations. Several of these Mendelian variants were only segregating in one Qatari subpopulation, where the observed subpopulation specificity trends were confirmed in an independent population of 386 Qataris. Premarital genetic screening in Qatar tests for only four out of the 37, such that this study provides a set of Mendelian disease variants with potential impact on the epidemiological profile of the population that could be incorporated into the testing program if further experimental and clinical characterization confirms high penetrance., (© 2013 WILEY PERIODICALS, INC.)

There is great scientific and popular interest in understanding the genetic history of populations in the Americas. We wish to understand when different regions of the continent were inhabited, where settlers came from, and how current inhabitants relate genetically to earlier populations. Recent studies unraveled parts of the genetic history of the continent using genotyping arrays and uniparental markers. The 1000 Genomes Project provides a unique opportunity for improving our understanding of population genetic history by providing over a hundred sequenced low coverage genomes and exomes from Colombian (CLM), Mexican-American (MXL), and Puerto Rican (PUR) populations. Here, we explore the genomic contributions of African, European, and especially Native American ancestry to these populations. Estimated Native American ancestry is 48% in MXL, 25% in CLM, and 13% in PUR. Native American ancestry in PUR is most closely related to populations surrounding the Orinoco River basin, confirming the Southern American ancestry of the Taíno people of the Caribbean. We present new methods to estimate the allele frequencies in the Native American fraction of the populations, and model their distribution using a demographic model for three ancestral Native American populations. These ancestral populations likely split in close succession: the most likely scenario, based on a peopling of the Americas 16 thousand years ago (kya), supports that the MXL Ancestors split 12.2kya, with a subsequent split of the ancestors to CLM and PUR 11.7kya. The model also features effective populations of 62,000 in Mexico, 8,700 in Colombia, and 1,900 in Puerto Rico. Modeling Identity-by-descent (IBD) and ancestry tract length, we show that post-contact populations also differ markedly in their effective sizes and migration patterns, with Puerto Rico showing the smallest effective size and the earlier migration from Europe. Finally, we compare IBD and ancestry assignments to find evidence for relatedness among European founders to the three populations., Competing Interests: The authors have declared that no competing interests exist.

Background: Populations of the Arabian Peninsula have a complex genetic structure that reflects waves of migrations including the earliest human migrations from Africa and eastern Asia, migrations along ancient civilization trading routes and colonization history of recent centuries., Results: Here, we present a study of genome-wide admixture in this region, using 156 genotyped individuals from Qatar, a country located at the crossroads of these migration patterns. Since haplotypes of these individuals could have originated from many different populations across the world, we have developed a machine learning method "SupportMix" to infer loci-specific genomic ancestry when simultaneously analyzing many possible ancestral populations. Simulations show that SupportMix is not only more accurate than other popular admixture discovery tools but is the first admixture inference method that can efficiently scale for simultaneous analysis of 50-100 putative ancestral populations while being independent of prior demographic information., Conclusions: By simultaneously using the 55 world populations from the Human Genome Diversity Panel, SupportMix was able to extract the fine-scale ancestry of the Qatar population, providing many new observations concerning the ancestry of the region. For example, as well as recapitulating the three major sub-populations in Qatar, composed of mainly Arabic, Persian, and African ancestry, SupportMix additionally identifies the specific ancestry of the Persian group to populations sampled in Greater Persia rather than from China and the ancestry of the African group to sub-Saharan origin and not Southern African Bantu origin as previously thought.

Background: The small airway epithelium (SAE), the cell population that covers the human airway surface from the 6th generation of airway branching to the alveoli, is the major site of lung disease caused by smoking. The focus of this study is to provide quantitative assessment of the SAE transcriptome in the resting state and in response to chronic cigarette smoking using massive parallel mRNA sequencing (RNA-Seq)., Results: The data demonstrate that 48% of SAE expressed genes are ubiquitous, shared with many tissues, with 52% enriched in this cell population. The most highly expressed gene, SCGB1A1, is characteristic of Clara cells, the cell type unique to the human SAE. Among other genes expressed by the SAE are those related to Clara cell differentiation, secretory mucosal defense, and mucociliary differentiation. The high sensitivity of RNA-Seq permitted quantification of gene expression related to infrequent cell populations such as neuroendocrine cells and epithelial stem/progenitor cells. Quantification of the absolute smoking-induced changes in SAE gene expression revealed that, compared to ubiquitous genes, more SAE-enriched genes responded to smoking with up-regulation, and those with the highest basal expression levels showed most dramatic changes. Smoking had no effect on SAE gene splicing, but was associated with a shift in molecular pattern from Clara cell-associated towards the mucus-secreting cell differentiation pathway with multiple features of cancer-associated molecular phenotype., Conclusions: These observations provide insights into the unique biology of human SAE by providing quantitative assessment of the global transcriptome under physiological conditions and in response to the stress of chronic cigarette smoking.

The Qatari population, located at the Arabian migration crossroads of African and Eurasia, is comprised of Bedouin, Persian and African genetic subgroups. By deep exome sequencing of only 7 Qataris, including individuals in each subgroup, we identified 2,750 nonsynonymous SNPs predicted to be deleterious, many of which are linked to human health, or are in genes linked to human health. Many of these SNPs were at significantly elevated deleterious allele frequency in Qataris compared to other populations worldwide. Despite the small sample size, SNP allele frequency was highly correlated with a larger Qatari sample. Together, the data demonstrate that exome sequencing of only a small number of individuals can reveal genetic variations with potential health consequences in understudied populations.

Background: Dopamine β-hydroxylase (DBH) plays an indispensable role in catecholamine synthesis by converting dopamine into norepinephrine. Here, we characterized a DBH promoter polymorphism (C-2073T; rs1989787; minor allele frequency ~16%) that influences not only gene transcription but also enzyme secretion and blood pressure (BP) in vivo., Methods: Plasma DBH activity was measured spectrophotometrically. DBH genetic effects on BP were tested in subjects with the most extreme BP values in a large primary care population. Functional effects of promoter variants were studied by site-directed mutagenesis in DBH promoter haplotype/luciferase reporter plasmids transfected into chromaffin cells. Sequence motifs were predicted from position weight matrices, and endogenous transcription factor binding was probed by Chromatin ImmunoPrecipitation (ChIP)., Results: The T-allele of common promoter variant C-2073T was contained in a promoter haplotype that associated with plasma DBH activity, a trait also predicted by that variant itself. Promoter haplotypes including C-2073T predicted BP in the population, and the effect was also referable to C-2073T itself. Computationally, C-2073 disrupted a predicted match for transcription factor c-FOS. Site-directed mutagenesis at C-2073T altered not only basal promoter activity, but also transactivation by c-FOS, as well as the chromaffin cell secretory stimuli nicotine or pituitary adenylate cyclase-activating polypeptide (PACAP). Endogenous c-FOS bound to the motif in chromatin., Conclusions: These results suggest that DBH promoter variant C-2073T is functional in vivo: this promoter variant seems to initiate a cascade of transcriptional and biochemical changes including augmented DBH secretion, eventuating in elevation of basal BP, and hence cardiovascular risk. The observations suggest new strategies for probing the pathophysiology, risk, and treatment of hypertension.

The catecholamine biosynthetic pathway consists of several enzymatic steps in series, beginning with the amino acids phenylalanine and tyrosine, and eventuating in the catecholamines norepinephrine (noradrenaline) and epinephrine (adrenaline). Since the enzyme tyrosine hydroxylase (TH; tyrosine 3-mono-oxygenase; EC 1.14.16.2; chromosome 11p15.5) is generally considered to be rate-limiting in this pathway, probed as to whether common genetic variation at the TH gene occurred, and whether such variants contributed to inter-individual alterations in autonomic function, either biochemical or physiological. We began with sequencing a tetranucleotide (TCAT) repeat in the first intron, and found that the two most common versions, (TCAT)(6) and (TCAT)(10i), predicted heritable autonomic traits in twin pairs. We then conducted systematic polymorphism discovery across the ~8 kbp locus, and discovered numerous variants, principally non-coding. The proximal promoter block contained four common variants, and its haplotypes and SNPs (especially C-824T, rs10770141) predicted catecholamine secretion, environmental stress-induced BP increments, and hypertension. Finally, we found that two of the common promoter variants, C-824T (rs10770141) and A-581G (rs10770140), were functional in that they differentially affected transcriptional activity of the isolated promoter, disrupted recognition motifs for specific transcription factor binding, altered the promoter responses to the co-transfected (exogenous) factors, and bound the endogenous factors in the chromatin fraction of the nucleus. We concluded that common variation in the proximal TH promoter is functional, giving rise to changes in autonomic function and consequently cardiovascular risk.

Objectives: The purpose of this study is to understand whether naturally occurring genetic variation in the promoter of chromogranin B (CHGB), a major constituent of catecholamine storage vesicles, is functional and confers risk for cardiovascular disease., Background: CHGB plays a necessary (catalytic) role in catecholamine storage vesicle biogenesis. Previously, we found that genetic variation at CHGB influenced autonomic function, with association maximal toward the 5' region., Methods: Here we explored transcriptional mechanisms of such effects, characterizing 2 common variants in the proximal promoter, A-296C and A-261T, using transfection/cotransfection, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP). We then tested the effects of promoter variation on cardiovascular traits., Results: The A-296C disrupted a c-FOS motif, exhibiting differential mobility shifting to chromaffin cell nuclear proteins during EMSA, binding of endogenous c-FOS on ChIP, and differential response to exogenous c-FOS. The A-261T disrupted motifs for SRY and YY1, with similar consequences for EMSA, endogenous factor binding, and responses to exogenous factors. The 2-SNP CHGB promoter haplotypes had a profound (p=3.16E-20) effect on blood pressure (BP) in the European ancestry population, with a rank order of CT

Background: Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. Common genetic variation at the human TH promoter predicts alterations in autonomic activity and blood pressure, but how such variation influences human traits and, specifically, whether such variation affects transcription are not yet known., Methods and Results: Pairwise linkage disequilibrium across the TH locus indicated that common promoter variants (C-824T, G-801C, A-581G, and G-494A) were located in a single 5' linkage disequilibrium block in white, black, Hispanic, and Asian populations. Polymorphisms C-824T and A-581G were located in highly conserved regions and were predicted to disrupt known transcriptional control motifs myocyte enhancer factor-2 (MEF2), sex-determining region Y (SRY), and forkhead box D1 (FOXD1) at C-824T and G/C-rich binding factors specificity protein 1 (SP1), activating enhancer-binding protein 2 (AP2)], early growth response protein 1 (EGR1) at A-581G. At C-824T and A-581G, promoter and luciferase reporter plasmids indicated differential allele strength (T>C at C-824T; G>A at A-581G) under both basal circumstances and secretory stimulation. C-824T and A-581G displayed the most pronounced effects on both transcription in cella and catecholamine secretion in vivo. We further probed the functional significance of C-824T and A-581G by cotransfection of trans-activating factors in cella; MEF2, SRY, and FOXD1 differentially activated C-824T, whereas the G/C-rich binding factors SP1, AP2, and EGR1 differentially activated A-581G. At C-824T, factor MEF2 acted in a directionally coordinate fashion (at T>C) to explain the in vivo trait associations, whereas at A-581G, factors SP1, AP2, and EGR1 displayed similar differential actions (at G>A). Finally, chromatin immunoprecipitation demonstrated that the endogenous factors bound to the motifs in cella., Conclusions: We conclude that common genetic variants in the proximal TH promoter, especially at C-824T and A-581G, are functional in cella and alter transcription so as to explain promoter marker-on-trait associations in vivo. MEF2, FOXD1, and SRY contribute to functional differences in C-824T expression, whereas SP1, AP2, and EGR1 mediate those of A-581G. The SRY effect on TH transcription suggests a mechanism whereby male and female sex may differ in sympathetic activity and hence blood pressure. These results point to new strategies for diagnostic and therapeutic intervention into disorders of human autonomic function and their cardiovascular consequences.

Processes underlying the formation of dense core secretory granules (DCGs) of neuroendocrine cells are poorly understood. Here, we present evidence that DCG biogenesis is dependent on the secretory protein secretogranin (Sg) II, a member of the granin family of pro-hormone cargo of DCGs in neuroendocrine cells. Depletion of SgII expression in PC12 cells leads to a decrease in both the number and size of DCGs and impairs DCG trafficking of other regulated hormones. Expression of SgII fusion proteins in a secretory-deficient PC12 variant rescues a regulated secretory pathway. SgII-containing dense core vesicles share morphological and physical properties with bona fide DCGs, are competent for regulated exocytosis, and maintain an acidic luminal pH through the V-type H(+)-translocating ATPase. The granulogenic activity of SgII requires a pH gradient along this secretory pathway. We conclude that SgII is a critical factor for the regulation of DCG biogenesis in neuroendocrine cells, mediating the formation of functional DCGs via its pH-dependent aggregation at the trans-Golgi network.

Chromogranin A (CgA), the major soluble protein in chromaffin granules, is proteolytically processed to generate biologically active peptides including the catecholamine release inhibitory peptide catestatin. Here we sought to determine whether cysteine protease cathepsin L (CTSL), a novel enzyme for proteolytic processing of neuropeptides, acts like the well-established serine proteases [prohormone convertase (PC)1/3 or PC2] to generate catestatin by proteolytic processing of CgA. We found that endogenous CTSL colocalizes with CgA in the secretory vesicles of primary rat chromaffin cells. Transfection of PC12 cells with an expression plasmid encoding CTSL directed expression of CTSL toward secretory vesicles. Deconvolution fluorescence microscopy suggested greater colocalization of CTSL with CgA than the lysosomal marker LGP110. The overexpression of CTSL in PC12 cells caused cleavage of full-length CgA. CTSL also cleaved CgA in vitro, in time- and dose-dependent fashion, and specificity of the process was documented through E64 (thiol reagent) inhibition. Mass spectrometry on CTSL-digested recombinant CgA identified a catestatin-region peptide, corresponding to CgA(360-373). The pool of peptides generated from the CTSL cleavage of CgA inhibited nicotine-induced catecholamine secretion from PC12 cells. CTSL processing in the catestatin region was diminished by naturally occurring catestatin variants, especially Pro370Leu and Gly364Ser. Among the CTSL-generated peptides, a subset matched those found in the catestatin region in vivo. These findings indicate that CgA can be a substrate for the cysteine protease CTSL both in vitro and in cella, and their colocalization within chromaffin granules in cella suggests the likelihood of an enzyme/substrate relationship in vivo.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. Does common genetic variation at human TH alter autonomic activity and predispose to cardiovascular disease? We undertook systematic polymorphism discovery at the TH locus, and then tested variants for contributions to sympathetic function and blood pressure. We resequenced 80 ethnically diverse individuals across the TH locus. One hundred seventy-two twin pairs were evaluated for sympathetic traits, including catecholamine production and environmental (cold) stress responses. To evaluate hypertension, we genotyped subjects selected from the most extreme diastolic blood pressure percentiles in the population. Human TH promoter haplotype/reporter plasmids were transfected into chromaffin cells. Forty-nine single nucleotide polymorphisms (SNPs) and one tetranucleotide repeat were discovered, but coding region polymorphism did not account for common phenotypic variation. A block of linkage disequilibrium spanned four common variants in the proximal promoter. Catecholamine secretory traits were significantly heritable, as were stress-induced blood pressure changes. In the TH promoter, significant associations were found for urinary catecholamine excretion, as well as blood pressure response to stress. TH promoter haplotype #2 (TGGG) showed pleiotropy, increasing both norepinephrine excretion and blood pressure during stress. In hypertension, a case-control study (1266 subjects, 53% women) established the effect of C-824T in determination of blood pressure. We conclude that human catecholamine secretory traits are heritable, displaying joint genetic determination (pleiotropy) with autonomic activity and finally with blood pressure in the population. Catecholamine secretion is influenced by genetic variation in the adrenergic pathway encoding catecholamine synthesis, especially at the classically rate-limiting step, TH. The results suggest novel pathophysiological links between a key adrenergic locus, catecholamine metabolism, and blood pressure, and suggest new strategies to approach the mechanism, diagnosis, and treatment of systemic hypertension.

Chromogranin A (CHGA) is stored and released from the same secretory vesicles that contain catecholamines in chromaffin cells and noradrenergic neurons. We had previously identified common genetic variants at the CHGA locus in several human populations. Here we focus on whether inter-individual variants in the promoter region are of physiological significance. A common haplotype, CGATA (Hap-B), blunted the blood pressure response to cold stress and the effect exhibited molecular heterosis with the greatest blood pressure change found in Hap-A/Hap-B heterozygotes. Homozygosity for three minor alleles with peak effects within the haplotype predicted lower stress-induced blood pressure changes. The G-462A variant predicted resting blood pressure in the population with higher pressures occurring in heterozygotes (heterosis). Using cells transfected with CHGA promoter-luciferase reporter constructs, the Hap-B haplotype had decreased luciferase expression compared to the TTGTC (Hap-A) haplotype under both basal conditions and after activation by pre-ganglionic stimuli. The G-462A variant altered a COUP-TF transcriptional control motif. The two alleles in transfected promoters differed in basal activity and in the responses to COUP-II-TF transactivation and to retinoic acid. In vitro findings of molecular heterosis were also noted with the transfected CHGA promoter wherein the diploid combination of the two G-462A alleles gave rise to higher luciferase expression than either allele in isolation. Our results suggest that common genetic variants in the CHGA promoter may regulate heritable changes in blood pressure.

GTP cyclohydrolase 1 (GCH1) is rate limiting in the provision of the cofactor tetrahydrobiopterin for biosynthesis of catecholamines and NO. We asked whether common genetic variation at GCH1 alters transmitter synthesis and predisposes to disease. Here we undertook a systematic search for polymorphisms in GCH1, then tested variants' contributions to NO and catecholamine release as well as autonomic function in twin pairs. Renal NO and neopterin excretions were significantly heritable, as were baroreceptor coupling (heart rate response to BP fluctuation) and pulse interval (1/heart rate). Common GCH1 variant C+243T in the 3'-untranslated region (3'-UTRs) predicted NO excretion, as well as autonomic traits: baroreceptor coupling, maximum pulse interval, and pulse interval variability, though not catecholamine secretion. In individuals with the most extreme BP values in the population, C+243T affected both diastolic and systolic BP, principally in females. In functional studies, C+243T decreased reporter expression in transfected 3'-UTRs plasmids. We conclude that human NO secretion traits are heritable, displaying joint genetic determination with autonomic activity by functional polymorphism at GCH1. Our results document novel pathophysiological links between a key biosynthetic locus and NO metabolism and suggest new strategies for approaching the mechanism, diagnosis, and treatment of risk predictors for cardiovascular diseases such as hypertension.

Background: Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. Does common genetic variation at human TH alter autonomic activity and predispose to cardiovascular disease? We undertook systematic polymorphism discovery at the TH locus and then tested variants for contributions to sympathetic function and blood pressure., Methods and Results: We resequenced 80 ethnically diverse individuals across the TH locus. One hundred seventy-two twin pairs were evaluated for sympathetic traits, including catecholamine production, reflex control of the circulation, and environmental (cold) stress responses. To evaluate hypertension, we genotyped subjects selected from the most extreme diastolic blood pressure percentiles in the population. Human TH promoter haplotype/reporter plasmids were transfected into chromaffin cells. Forty-nine single-nucleotide polymorphisms were discovered, but coding region polymorphism did not account for common phenotypic variation. A block of linkage disequilibrium spanned 4 common variants in the proximal promoter. Catecholamine secretory traits were significantly heritable (h2), as were stress-induced blood pressure changes. In the TH promoter, significant associations were found for urinary catecholamine excretion and for blood pressure response to stress. TH promoter haplotype 2 (TGGG) showed pleiotropy, increasing both norepinephrine excretion and blood pressure during stress. Coalescent simulations suggest that TH haplotype 2 likely arose approximately 380,000 years ago. In hypertension, 2 independent case-control studies (1266 subjects with 53% women and 927 subjects with 24% women) replicated the effect of C-824T in the determination of blood pressure., Conclusions: We conclude that human catecholamine secretory traits are heritable, displaying joint genetic determination (pleiotropy) with autonomic activity and finally with blood pressure in the population. Catecholamine secretion is influenced by genetic variation in the adrenergic pathway encoding catecholamine synthesis, especially at the classically rate-limiting step, TH. The results suggest novel pathophysiological links between a key adrenergic locus, catecholamine metabolism, and blood pressure and suggest new strategies to approach the mechanism, diagnosis, and treatment of systemic hypertension.