Copper Oxide Nanoparticles Induced Growth and Physio-Biochemical Changes in Maize (Zea mays L.) in Saline Soil.

Research on nanoparticles (NPs) is gaining great attention in modulating abiotic stress tolerance and improving crop productivity. Therefore, this investigation was carried out to evaluate the effects of copper oxide nanoparticles (CuO-NPs) on growth and biochemical characteristics in two maize hybrids (YH-5427 and FH-1046) grown under normal conditions or subjected to saline stress. A pot-culture experiment was carried out in the Botanical Research Area of "the University of Lahore", Lahore, Pakistan, in a completely randomized design. At two phenological stages, both maize hybrids were irrigated with the same amount of distilled water or NaCl solution (EC = 5 dS m-1) and subjected or not to foliar treatment with a suspension of CuO-NPs. The salt stress significantly reduced the photosynthetic parameters (photosynthetic rate, transpiration, stomatal conductance), while the sodium content in the shoot and root increased. The foliar spray with CuO-NPs improved the growth and photosynthetic attributes, along with the N, P, K, Ca, and Mg content in the roots and shoots. However, the maize hybrid YH-5427 responded better than the other hybrid to the saline stress when sprayed with CuO-NPs. Overall, the findings of the current investigation demonstrated that CuO-NPs can help to reduce the adverse effects of salinity stress on maize plants by improving growth and physio-biochemical attributes.

Lead Toxicity-Mediated Growth and Metabolic Alterations at Early Seedling Stages of Maize (Zea mays L.).

To investigate the toxic effects of lead (Pb) on key metabolic activities essential for proper germination and seedling growth of maize seeds, experiments were carried out with different levels of Pb (0 to 120 mg of Pb L-1 as PbCl2) applied through growth medium to two maize hybrids H-3310S and H-6724. The research findings indicated that growth and metabolic activities were adversely affected by increased Pb contamination in growth medium; however, a slow increase in these parameters was recorded with increasing time from 0 to 120 h. Protease activity decreased with an increase in the level of Pb contamination but increased with time; consequently, a reduction in seed proteins and an increase in total free amino acids were observed with time. Similarly, α-amylase activity decreased with an increase in Pb concentration in growth medium while it increased with increasing time from 0 to 120 h; consequently, reducing and non-reducing sugars increased with time but decreased with exposure to lead. The roots of both maize hybrids had higher Pb contents than those of the shoot, which decreased the uptake of nitrogen, phosphorus, and potassium. All these nutrients are essential for optimal plant growth; therefore, the reduction in growth and biomass of maize seedlings could be due to Pb toxicity that altered metabolic processes, as sugar and amino acids are necessary for the synthesis of metabolic compounds, rapid cell division, and proper functioning of enzymes in the growing embryo, but all were dramatically reduced due to suppression of protease and α-amylase by toxicity of Pb. In general, hybrid H-3310S performed better in Pb-contaminated growth medium than H-6724.

Mass-spectrometric analysis of APOB polymorphism rs1042031 (G/T) and its influence on serum proteome of coronary artery disease patients: genetic-derived proteomics consequences.

Genetic polymorphisms of apolipoprotein B gene (APOB) may result into serum proteomic perturbance in Coronary Artery Disease (CAD). The current case-control cohort of Pakistani subjects was designed to analyze the genetic influence of APOB rs1042031, (G/T) genotype on serum proteome. Subjects were categorized into two groups: CAD patients (n = 480) and healthy individuals (n = 220). For genotyping, tetra ARMS-PCR was carried out and validated through sequencing, whereas LC/MS-based proteomic analysis of serum samples was performed through label-free quantification. In initial step of genotyping, the frequencies of each genotype GG, GT, and TT were 70%, 27%, and 30% in CAD patients, while in control group, the subjects were 52%, 43%, and 5%, respectively, in CAD patients. The genotypic frequencies in patients vs. control groups found significantly different (p = 0.004), and a strong association of dominant alleles GG with the CAD was observed in both dominant (OR: 2.4 (1.71-3.34), p = 0.001) and allelic genetic models (OR: 2.0 (1.45-2.86), p = 0.001). In second step of label-free quantitation, a total of 40 significant proteins were found with altered expression in CAD patients. The enriched Gene Ontology (GO) terms of molecular functions and pathways of these protein showed upregulated pathways as follows: chylomicron remodeling and assembly, complement cascade activation, plasma lipoprotein assembly, apolipoprotein-A receptor binding, and metabolism of fat-soluble vitamins in G allele carrier of rs1042031 (G > T) vs. mutant T-allele carriers. This study provides better understanding of CAD pathobiology by proteogenomics of APOB. It evidences the influence of APOB rs1042031-dominant (GG) genotype with CAD patients.

South Asian medical cohorts reveal strong founder effects and high rates of homozygosity.

The benefits of large-scale genetic studies for healthcare of the populations studied are well documented, but these genetic studies have traditionally ignored people from some parts of the world, such as South Asia. Here we describe whole genome sequence (WGS) data from 4806 individuals recruited from the healthcare delivery systems of Pakistan, India and Bangladesh, combined with WGS from 927 individuals from isolated South Asian populations. We characterize population structure in South Asia and describe a genotyping array (SARGAM) and imputation reference panel that are optimized for South Asian genomes. We find evidence for high rates of reproductive isolation, endogamy and consanguinity that vary across the subcontinent and that lead to levels of rare homozygotes that reach 100 times that seen in outbred populations. Founder effects increase the power to associate functional variants with disease processes and make South Asia a uniquely powerful place for population-scale genetic studies.

Tetra-ARMS-PCR assay development for genotyping of AGT rs699 T/C polymorphism, its comparison with PCR-RFLP and application in a case-control association study of cardiovascular disease patients.

Genetic variations in the AGT gene play a significant role in controlling the plasma concentration of angiotensinogen (precursor protein of bioactive octapeptide angiotensin II) and the efficacy of antihypertensive drugs. In the current study, Tetra-Amplification Refractory Mutation System-Polymerase Chain Reaction (T-ARMS-PCR) was developed for genotyping of AGT rs699 T/C polymorphism and validated through Sanger DNA sequencing. Its efficiency was also tested using 474 human DNA samples [control, n = 181; cardiovascular disease (CVD) patients, n = 293]. Results showed that T-ARMS-PCR is superior to the commonly used PCR-Restriction Fragment Length Polymorphism (PCR-RFLP). Statistical analysis revealed that the AGT rs699 CC genotype is more prevalent in the CVD patient group (37% vs. 28%) and AGT rs699 C allele and CC genotype increased the risk of CVD by 1.4 and 1.9 fold, respectively. In summary, T-ARMS-PCR is the most suitable approach for quick and efficient genotyping of AGT rs699 T/C polymorphism in a large population in resource-limited countries, Furthermore, AGT rs699 T/C polymorphism is associated with the risk of CVD in the Punjabi Pakistani population.

Association of vitamin D receptor polymorphisms with cardiometabolic conditions in Pakistani population.

Apart from bone related effects, vitamin D has roles in immune modulation, hypertension, diabetes and cardiovascular diseases. Metabolic functions of vitamin D are mediated after binding with vitamin D receptor (VDR). VDR polymorphisms affect its physiological functions. Several VDR single nucleotide polymorphisms (SNPs) are reported previously. However, VDR polymorphisms causing influence on cardiovascular and metabolic disorders have not been investigated in Pakistani population so far. Therefore, present study was conducted to evaluate the role of VDR polymorphisms (rs2228570 and rs7975232) in the pathobiology of cardiometabolic disorders. In all, 400 cardiometabolic patients and 226 healthy control human adults were enrolled from Faisalabad, Pakistan. Biochemical parameters (serum glucose, liver function test, renal function test and lipid profile) were analyzed by standard kit methods. Genetic analysis was done by ARMS-PCR assay. Data was analyzed in SPSS v20. Regression analysis revealed that GG and AG genotypes of rs2228570 A>G polymorphism significantly increased the risk of hypertension in cardiovascular patients by 5.29 and 5.94 times respectively (GG: OR=5.29, 95% CI=1.63-17.2, p=0.005; AG: OR=5.94, 95% CI=1.70-20.7, p=0.005). However, rs7975232 C>A polymorphism was not correlated with cardiometabolic conditions. In conclusion, GG and AG genotypes of VDR SNP rs2228570 significantly contribute for hypertension in cardiovascular disease patients.

Analyzing human knockouts to validate GPR151 as a therapeutic target for reduction of body mass index.

Novel drug targets for sustained reduction in body mass index (BMI) are needed to curb the epidemic of obesity, which affects 650 million individuals worldwide and is a causal driver of cardiovascular and metabolic disease and mortality. Previous studies reported that the Arg95Ter nonsense variant of GPR151, an orphan G protein-coupled receptor, is associated with reduced BMI and reduced risk of Type 2 Diabetes (T2D). Here, we further investigate GPR151 with the Pakistan Genome Resource (PGR), which is one of the largest exome biobanks of human homozygous loss-of-function carriers (knockouts) in the world. Among PGR participants, we identify eleven GPR151 putative loss-of-function (plof) variants, three of which are present at homozygosity (Arg95Ter, Tyr99Ter, and Phe175LeufsTer7), with a cumulative allele frequency of 2.2%. We confirm these alleles in vitro as loss-of-function. We test if GPR151 plof is associated with BMI, T2D, or other metabolic traits and find that GPR151 deficiency in complete human knockouts is not associated with clinically significant differences in these traits. Relative to Gpr151+/+ mice, Gpr151-/- animals exhibit no difference in body weight on normal chow and higher body weight on a high-fat diet. Together, our findings indicate that GPR151 antagonism is not a compelling therapeutic approach to treatment of obesity.

Blockchain Based Authentication and Cluster Head Selection Using DDR-LEACH in Internet of Sensor Things.

This paper proposes a blockchain-based node authentication model for the Internet of sensor things (IoST). The nodes in the network are authenticated based on their credentials to make the network free from malicious nodes. In IoST, sensor nodes gather the information from the environment and send it to the cluster heads (CHs) for additional processing. CHs aggregate the sensed information. Therefore, their energy rapidly depletes due to extra workload. To solve this issue, we proposed distance, degree, and residual energy-based low-energy adaptive clustering hierarchy (DDR-LEACH) protocol. DDR-LEACH is used to replace CHs with the ordinary nodes based on maximum residual energy, degree, and minimum distance from BS. Furthermore, storing a huge amount of data in the blockchain is very costly. To tackle this issue, an external data storage, named as interplanetary file system (IPFS), is used. Furthermore, for ensuring data security in IPFS, AES 128-bit is used, which performs better than the existing encryption schemes. Moreover, a huge computational cost is required using a proof of work consensus mechanism to validate transactions. To solve this issue, proof of authority (PoA) consensus mechanism is used in the proposed model. The simulation results are carried out, which show the efficiency and effectiveness of the proposed system model. The DDR-LEACH is compared with LEACH and the simulation results show that DDR-LEACH outperforms LEACH in terms of energy consumption, throughput, and improvement in network lifetime with CH selection mechanism. Moreover, transaction cost is computed, which is reduced by PoA during data storage on IPFS and service provisioning. Furthermore, the time is calculated in the comparison of AES 128-bit scheme with existing scheme. The formal security analysis is performed to check the effectiveness of smart contract against attacks. Additionally, two different attacks, MITM and Sybil, are induced in our system to show our system model's resilience against cyber attacks.

Effect of APOB polymorphism rs562338 (G/A) on serum proteome of coronary artery disease patients: a "proteogenomic" approach.

In the current study, APOB (rs1052031) genotype-guided proteomic analysis was performed in a cohort of Pakistani population. A total of 700 study subjects, including Coronary Artery Disease (CAD) patients (n = 480) and healthy individuals (n = 220) as a control group were included in the study. Genotyping was carried out by using tetra primer-amplification refractory mutation system-based polymerase chain reaction (T-ARMS-PCR) whereas mass spectrometry (Orbitrap MS) was used for label free quantification of serum samples. Genotypic frequency of GG genotype was found to be 90.1%, while 6.4% was for GA genotype and 3.5% was for AA genotypes in CAD patients. In the control group, 87.2% healthy subjects were found to have GG genotype, 11.8% had GA genotype, and 0.9% were with AA genotypes. Significant (p = 0.007) difference was observed between genotypic frequencies in the patients and the control group. The rare allele AA was found to be strongly associated with the CAD [OR: 4 (1.9-16.7)], as compared to the control group in recessive genetic model (p = 0.04). Using label free proteomics, altered expression of 60 significant proteins was observed. Enrichment analysis of these protein showed higher number of up-regulated pathways, including phosphatidylcholine-sterol O-acyltransferase activator activity, cholesterol transfer activity, and sterol transfer activity in AA genotype of rs562338 (G>A) as compared to the wild type GG genotype. This study provides a deeper insight into CAD pathobiology with reference to proteogenomics, and proving this approach as a good platform for identifying the novel proteins and signaling pathways in relation to cardiovascular diseases.

Genome-wide analysis of blood lipid metabolites in over 5000 South Asians reveals biological insights at cardiometabolic disease loci.

BACKGROUND: Genetic, lifestyle, and environmental factors can lead to perturbations in circulating lipid levels and increase the risk of cardiovascular and metabolic diseases. However, how changes in individual lipid species contribute to disease risk is often unclear. Moreover, little is known about the role of lipids on cardiovascular disease in Pakistan, a population historically underrepresented in cardiovascular studies. METHODS: We characterised the genetic architecture of the human blood lipidome in 5662 hospital controls from the Pakistan Risk of Myocardial Infarction Study (PROMIS) and 13,814 healthy British blood donors from the INTERVAL study. We applied a candidate causal gene prioritisation tool to link the genetic variants associated with each lipid to the most likely causal genes, and Gaussian Graphical Modelling network analysis to identify and illustrate relationships between lipids and genetic loci. RESULTS: We identified 253 genetic associations with 181 lipids measured using direct infusion high-resolution mass spectrometry in PROMIS, and 502 genetic associations with 244 lipids in INTERVAL. Our analyses revealed new biological insights at genetic loci associated with cardiometabolic diseases, including novel lipid associations at the LPL, MBOAT7, LIPC, APOE-C1-C2-C4, SGPP1, and SPTLC3 loci. CONCLUSIONS: Our findings, generated using a distinctive lipidomics platform in an understudied South Asian population, strengthen and expand the knowledge base of the genetic determinants of lipids and their association with cardiometabolic disease-related loci.

Outcome of thrombolysis with streptokinase in patients with prosthetic valve thrombosis.

OBJECTIVE: To evaluate the outcome of thrombolysis in patients of prosthetic valve thrombosis. METHODS: This retrospective analysis was conducted on data of 84 patients of prosthetic valve thrombosis who presented to emergency room of Faisalabad Institute of Cardiology between July 2017 to December 2019. The diagnosis of prosthetic valve thrombosis was based on clinical suspicion and bed side transthoracic echocardiography done by a consultant cardiologist. Fluoroscopy was done to confirm the diagnosis by observing immobile valve leaflet. The confirmed patients were then treated in emergency with streptokinase after taking an informed consent. Quantitative variables like age were summarized by mean and standard deviation. Qualitative variables like gender, successful thrombolysis, stroke, major bleeding, mortality or re-do surgery were summarized by frequency and percentage. RESULTS: Mean age was 29 ± 6.36, years and there were more female patients (n=43, 51.25%) as compared to males (n=41, 48.8%). Among the 66 surviving patients thrombolysis was successful without any complications in 56 patients (66.7%). Thrombolysis was successful with minor complications in six patients (7.1%) and it failed to produce desired results in four patients (4.8%). In this study 18 (21.4%) patients died. The common complications included minor bleeding in four patients (4.8%) and major bleeding in 10 patients (12.0 %). CONCLUSION: Thrombolysis produces reasonable success rate in cases of prosthetic valve thrombosis who are in functional class I or II. However, it has very high mortality rate in patients presenting with functional class III and IV.

Discovery of 318 new risk loci for type 2 diabetes and related vascular outcomes among 1.4 million participants in a multi-ancestry meta-analysis.

We investigated type 2 diabetes (T2D) genetic susceptibility via multi-ancestry meta-analysis of 228,499 cases and 1,178,783 controls in the Million Veteran Program (MVP), DIAMANTE, Biobank Japan and other studies. We report 568 associations, including 286 autosomal, 7 X-chromosomal and 25 identified in ancestry-specific analyses that were previously unreported. Transcriptome-wide association analysis detected 3,568 T2D associations with genetically predicted gene expression in 687 novel genes; of these, 54 are known to interact with FDA-approved drugs. A polygenic risk score (PRS) was strongly associated with increased risk of T2D-related retinopathy and modestly associated with chronic kidney disease (CKD), peripheral artery disease (PAD) and neuropathy. We investigated the genetic etiology of T2D-related vascular outcomes in the MVP and observed statistical SNP-T2D interactions at 13 variants, including coronary heart disease (CHD), CKD, PAD and neuropathy. These findings may help to identify potential therapeutic targets for T2D and genomic pathways that link T2D to vascular outcomes.

Association of ACE I/D Polymorphism with Risk of Diabetes in Cardiovascular Disease Patients.

Angiotensin converting enzyme (ACE), as part of renin angiotensin aldosterone system, is involved in blood pressure regulation and control several physiological functions. Insertion/Deletion (I/D) polymorphism of ACE has pronounced effects on development of metabolic diseases like diabetes, cardiovascular diseases (CVDs) and hypertension. However, association of I/D polymorphism with risk of diabetes in CVD patients is not known. The aim of present study was to check the association of ACE I/D polymorphism with risk of diabetes in subjects with CVD. For this, 531 subjects were sampled and divided into 3 groups; G1-H: (healthy controls, n=117), G2-CN: (cardiac patients without diabetes, n=271) and G3-CD: (cardiac patients with diabetes, n=143). Genotyping of ACE I/D polymorphism was done by polymerase chain reaction. Allelic and genotypic frequencies were in Hardy Weinberg Equilibrium (χ2=0.11, p>0.05) and revealed high prevalence of I allele (55%) among all groups. However, II genotype was more common (37%) in G3-CD: group. Level of glucose was also higher in subjects with II genotype than DD genotype (12.6±6.3 mmol/L vs. 9.7±5.1 mmol/L). Logistic regression analysis revealed that ACE II genotype increase the risk of diabetes in CVD patients by ~2 times [OR=1.94, CI: 1.24-3.01, p=0.03]; however, this association did not reach the significance level when adjusted for age and gender. In conclusion, ACE I/D polymorphism influence the risk of diabetes in CVD patients and ACE II increases this risk by ~2 fold.

The effect of oral progesterone for the treatment of abnormal uterine bleeding in women taking warfarin following prosthetic valve replacement.

OBJECTIVES: To evaluate the effect of oral progesterone for the treatment of abnormal uterine bleeding in patients taking warfarin after prosthetic valve replacement. METHODS: A total of 85 women of reproductive age, who were on warfarin due to prosthetic valve replacement were enrolled in the study. After detailed evaluation, their menstrual bleeding was quantified using Pictorial Bleeding Evaluation Chart. The patients were then prescribed an oral progesterone (Norethisterone) 5mg three times daily. The first follow up was done after one-month then at 3-months and at six months. The improvement in PBAC score was recorded at each visit. Data was entered and analyzed using SPSS (version 23.0). The mean ± Standard Deviation were calculated for quantitative variables while qualitative variables were presented in frequency table. The normality of data was checked using Kolmogorov-Smirnov test. Due lack of normal distribution of data in various groups, the Wilcoxon Sign Rank test was used to test the significance before and after treatment. The p-value of <0.05 was taken as statistically significant. RESULTS: The mean age of the patients was 30.13±7.69 years. The mean PBAC score was 162.8 ± 24.86 before initiation of treatment while at the end of the treatment it was 105.48 ± 8.38. Forty-six (54.1%) patients had continuous per vaginal bleeding, 33 (38.8%) had menorrhagia, 4 (4.7%) had inter-menstrual bleeding and 2 (2.4%) had menorrhagia along with polymenorrhea. The mean dose of warfarin taken by the patients was 5.85 ± 2.69 mg. The median parity of the patients was 2. The Wilcoxon Sign Rank test showed p-value of <0.00001 for comparison of the pre-treatment PCBA values with those of one, three and six-months after the treatment. The Friedman's test also had a p-value of <0.00001. This confirmed that the post treatment bleeding was significantly less than pretreatment bleeding. CONCLUSION: The warfarin induced abnormal uterine bleeding can be controlled effectively and safely with low dose of oral progesterone.

An Unbiased Lipid Phenotyping Approach To Study the Genetic Determinants of Lipids and Their Association with Coronary Heart Disease Risk Factors.

Direct infusion high-resolution mass spectrometry (DIHRMS) is a novel, high-throughput approach to rapidly and accurately profile hundreds of lipids in human serum without prior chromatography, facilitating in-depth lipid phenotyping for large epidemiological studies to reveal the detailed associations of individual lipids with coronary heart disease (CHD) risk factors. Intact lipid profiling by DIHRMS was performed on 5662 serum samples from healthy participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS). We developed a novel semi-targeted peak-picking algorithm to detect mass-to-charge ratios in positive and negative ionization modes. We analyzed lipid partial correlations, assessed the association of lipid principal components with established CHD risk factors and genetic variants, and examined differences between lipids for a common genetic polymorphism. The DIHRMS method provided information on 360 lipids (including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, and sterol lipids), with a median coefficient of variation of 11.6% (range: 5.4-51.9). The lipids were highly correlated and exhibited a range of associations with clinical chemistry biomarkers and lifestyle factors. This platform can provide many novel insights into the effects of physiology and lifestyle on lipid metabolism, genetic determinants of lipids, and the relationship between individual lipids and CHD risk factors.

Sensitivity to house dust mite allergens and prevalence of allergy-causing house dust mite species in Pothwar, Pakistan.

This study is the first report on the epidemiological status of house dust mite (HDM) allergy in Pothwar, Pakistan. Allergy data of 2087 symptomatic patients were obtained, of whom 1706 (81.7%) patients were skin-prick-test positive for HDM allergens. This percentage was significantly higher than for pollen and food allergens. In the results of this study Dermatophagoides farinae (61%) and D. pteronyssinus (29%) were the predominant species in the study area. Besides these pyroglyphids, predatory Cheyletus sp. (10%) and an oribatid mite sp. (1%) were also observed. Random and patients' houses showed 87.4 and 87.1% positive mite infestation, respectively. Mean (± SEM) D. farinae counts per g of dust in random samples was 235.4 ± 7.93 compared to 274.7 ± 10.78 from patients' homes. Mean D. pteronyssinus counts from random houses compared to patients' houses were 115.0 ± 4.57 and 124.6 ± 5.76, respectively. Mite counts depicted seasonal variation, with peaks during monsoon season. ELISA results of dust samples demonstrated that of the dust samples with > 10 µg/g of dust, the threshold value described as a risk factor for developing asthma, 57.6% had Der f1 and 20% Der p1 allergen load. Mean Der f1 burden was significantly higher than Der p1, with maximum levels during monsoon and autumn seasons. This research established a better awareness about the epidemiological status of HDM allergy and prevalence of allergy causing HDM species in Pakistan.

Identification of new susceptibility loci for type 2 diabetes and shared etiological pathways with coronary heart disease.

To evaluate the shared genetic etiology of type 2 diabetes (T2D) and coronary heart disease (CHD), we conducted a genome-wide, multi-ancestry study of genetic variation for both diseases in up to 265,678 subjects for T2D and 260,365 subjects for CHD. We identify 16 previously unreported loci for T2D and 1 locus for CHD, including a new T2D association at a missense variant in HLA-DRB5 (odds ratio (OR) = 1.29). We show that genetically mediated increase in T2D risk also confers higher CHD risk. Joint T2D-CHD analysis identified eight variants-two of which are coding-where T2D and CHD associations appear to colocalize, including a new joint T2D-CHD association at the CCDC92 locus that also replicated for T2D. The variants associated with both outcomes implicate new pathways as well as targets of existing drugs, including icosapent ethyl and adipocyte fatty-acid-binding protein.

Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity.

A major goal of biomedicine is to understand the function of every gene in the human genome. Loss-of-function mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such 'human knockouts' can provide insight into gene function. Consanguineous unions are more likely to result in offspring carrying homozygous loss-of-function mutations. In Pakistan, consanguinity rates are notably high. Here we sequence the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS), designed to understand the determinants of cardiometabolic diseases in individuals from South Asia. We identified individuals carrying homozygous predicted loss-of-function (pLoF) mutations, and performed phenotypic analysis involving more than 200 biochemical and disease traits. We enumerated 49,138 rare (<1% minor allele frequency) pLoF mutations. These pLoF mutations are estimated to knock out 1,317 genes, each in at least one participant. Homozygosity for pLoF mutations at PLA2G7 was associated with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; at TREH, with lower concentrations of apoB-containing lipoprotein subfractions; at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations; and at SLC9A3R1, with mediators of calcium and phosphate signalling. Heterozygous deficiency of APOC3 has been shown to protect against coronary heart disease; we identified APOC3 homozygous pLoF carriers in our cohort. We recruited these human knockouts and challenged them with an oral fat load. Compared with family members lacking the mutation, individuals with APOC3 knocked out displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Overall, these observations provide a roadmap for a 'human knockout project', a systematic effort to understand the phenotypic consequences of complete disruption of genes in humans.

Apolipoprotein(a) isoform size, lipoprotein(a) concentration, and coronary artery disease: a mendelian randomisation analysis.

BACKGROUND: The lipoprotein(a) pathway is a causal factor in coronary heart disease. We used a genetic approach to distinguish the relevance of two distinct components of this pathway, apolipoprotein(a) isoform size and circulating lipoprotein(a) concentration, to coronary heart disease. METHODS: In this mendelian randomisation study, we measured lipoprotein(a) concentration and determined apolipoprotein(a) isoform size with a genetic method (kringle IV type 2 [KIV2] repeats in the LPA gene) and a serum-based electrophoretic assay in patients and controls (frequency matched for age and sex) from the Pakistan Risk of Myocardial Infarction Study (PROMIS). We calculated odds ratios (ORs) for myocardial infarction per 1-SD difference in either LPA KIV2 repeats or lipoprotein(a) concentration. In a genome-wide analysis of up to 17 503 participants in PROMIS, we identified genetic variants associated with either apolipoprotein(a) isoform size or lipoprotein(a) concentration. Using a mendelian randomisation study design and genetic data on 60 801 patients with coronary heart disease and 123 504 controls from the CARDIoGRAMplusC4D consortium, we calculated ORs for myocardial infarction with variants that produced similar differences in either apolipoprotein(a) isoform size in serum or lipoprotein(a) concentration. Finally, we compared phenotypic versus genotypic ORs to estimate whether apolipoprotein(a) isoform size, lipoprotein(a) concentration, or both were causally associated with coronary heart disease. FINDINGS: The PROMIS cohort included 9015 patients with acute myocardial infarction and 8629 matched controls. In participants for whom KIV2 repeat and lipoprotein(a) data were available, the OR for myocardial infarction was 0·93 (95% CI 0·90-0·97; p<0·0001) per 1-SD increment in LPA KIV2 repeats after adjustment for lipoprotein(a) concentration and conventional lipid concentrations. The OR for myocardial infarction was 1·10 (1·05-1·14; p<0·0001) per 1-SD increment in lipoprotein(a) concentration, after adjustment for LPA KIV2 repeats and conventional lipids. Genome-wide analysis identified rs2457564 as a variant associated with smaller apolipoprotein(a) isoform size, but not lipoprotein(a) concentration, and rs3777392 as a variant associated with lipoprotein(a) concentration, but not apolipoprotein(a) isoform size. In 60 801 patients with coronary heart disease and 123 504 controls, OR for myocardial infarction was 0·96 (0·94-0·98; p<0·0001) per 1-SD increment in apolipoprotein(a) protein isoform size in serum due to rs2457564, which was directionally concordant with the OR observed in PROMIS for a similar change. The OR for myocardial infarction was 1·27 (1·07-1·50; p=0·007) per 1-SD increment in lipoprotein(a) concentration due to rs3777392, which was directionally concordant with the OR observed for a similar change in PROMIS. INTERPRETATION: Human genetic data suggest that both smaller apolipoprotein(a) isoform size and increased lipoprotein(a) concentration are independent and causal risk factors for coronary heart disease. Lipoprotein(a)-lowering interventions could be preferentially effective in reducing the risk of coronary heart disease in individuals with smaller apolipoprotein(a) isoforms. FUNDING: British Heart Foundation, US National Institutes of Health, Fogarty International Center, Wellcome Trust, UK Medical Research Council, UK National Institute for Health Research, and Pfizer.

Prevalence of sensitization to food allergens and challenge proven food allergy in patients visiting allergy centers in Rawalpindi and Islamabad, Pakistan.

In this study, we estimated the prevalence of food allergy in the adult allergic patients of Rawalpindi and Islamabad , Pakistan, based on self-report, skin prick test (SPT) and oral food challenge test (OFC). SPT was used for the estimation of sensitization to wheat, egg, milk, beef, chicken, mutton, fish, corn, lentils, rice, soya, peanut and banana. Among 689 patients, 39.19 % showed sensitivity to one or more foods, where, sensitization to wheat (156; 22.6 %) was highest, followed by egg (148; 21.48 %) and milk (138; 20.03 %). Sensitization to various proteins ranged between 15.53-15.97 %, while lentils, corn, rice, soya and peanut sensitization was 15.4, 16, 12.5, 12 and 11.5 % respectively. Only 7.1 % patients were SPT positive for banana allergen. SPT was performed in patients with self-reported food allergy (341/689) and also with no self-reported history of food allergy (348/689). SPT results were positive in 69.8 % of the self-report group, whereas, in the patients with no self-reported food allergy 9.2 % were found sensitized to one or more tested food allergens. 101 patients were recruited for OFC, 61 % of these were confirmed of food allergy. The prevalence of food allergy in the study population was 9 %. Food specific OFC results show that wheat allergy is affecting 1.6 % (95 % CI 0.9-2.84 %) of the total allergy patients, followed by egg allergy 1.31 % (95 % CI 0.70-2.47 %). Furthermore, corn allergy, rice allergy and peanut allergy were 1.02, 0.87 and 0.73 %, respectively. In conclusion, wheat allergy is the most prevalent, followed by egg, chicken, beef and fish allergy, respectively.