Whole-exome sequencing reveals damaging gene variants associated with hypoalphalipoproteinemia.

Low levels of high density lipoprotein-cholesterol (HDL-C) are associated with an elevated risk of arteriosclerotic coronary heart disease. Heritability of HDL-C levels is high. In this research discovery study, we used whole-exome sequencing to identify damaging gene variants that may play significant roles in determining HDL-C levels. We studied 204 individuals with a mean HDL-C level of 27.8 ± 6.4 mg/dl (range: 4-36 mg/dl). Data were analyzed by statistical gene burden testing and by filtering against candidate gene lists. We found 120 occurrences of probably damaging variants (116 heterozygous; four homozygous) among 45 of 104 recognized HDL candidate genes. Those with the highest prevalence of damaging variants were ABCA1 (n = 20), STAB1 (n = 9), OSBPL1A (n = 8), CPS1 (n = 8), CD36 (n = 7), LRP1 (n = 6), ABCA8 (n = 6), GOT2 (n = 5), AMPD3 (n = 5), WWOX (n = 4), and IRS1 (n = 4). Binomial analysis for damaging missense or loss-of-function variants identified the ABCA1 and LDLR genes at genome-wide significance. In conclusion, whole-exome sequencing of individuals with low HDL-C showed the burden of damaging rare variants in the ABCA1 and LDLR genes is particularly high and revealed numerous occurrences in HDL candidate genes, including many genes identified in genome-wide association study reports. Many of these genes are involved in cancer biology, which accords with epidemiologic findings of the association of HDL deficiency with increased risk of cancer, thus presenting a new area of interest in HDL genomics.

Towards a reference genome that captures global genetic diversity.

The current human reference genome is predominantly derived from a single individual and it does not adequately reflect human genetic diversity. Here, we analyze 338 high-quality human assemblies of genetically divergent human populations to identify missing sequences in the human reference genome with breakpoint resolution. We identify 127,727 recurrent non-reference unique insertions spanning 18,048,877 bp, some of which disrupt exons and known regulatory elements. To improve genome annotations, we linearly integrate these sequences into the chromosomal assemblies and construct a Human Diversity Reference. Leveraging this reference, an average of 402,573 previously unmapped reads can be recovered for a given genome sequenced to ~40X coverage. Transcriptomic diversity among these non-reference sequences can also be directly assessed. We successfully map tens of thousands of previously discarded RNA-Seq reads to this reference and identify transcription evidence in 4781 gene loci, underlining the importance of these non-reference sequences in functional genomics. Our extensive datasets are important advances toward a comprehensive reference representation of global human genetic diversity.

Three patients with homozygous familial hypercholesterolemia: Genomic sequencing and kindred analysis.

BACKGROUND: Homozygous Familial Hypercholesterolemia (HoFH) is an inherited recessive condition associated with extremely high levels of low-density lipoprotein (LDL) cholesterol in affected individuals. It is usually caused by homozygous or compound heterozygous functional mutations in the LDL receptor (LDLR). A number of mutations causing FH have been reported in literature and such genetic heterogeneity presents great challenges for disease diagnosis. OBJECTIVE: We aim to determine the likely genetic defects responsible for three cases of pediatric HoFH in two kindreds. METHODS: We applied whole exome sequencing (WES) on the two probands to determine the likely functional variants among candidate FH genes. We additionally applied 10x Genomics (10xG) Linked-Reads whole genome sequencing (WGS) on one of the kindreds to identify potentially deleterious structural variants (SVs) underlying HoFH. A PCR-based screening assay was also established to detect the LDLR structural variant in a cohort of 641 patients with elevated LDL. RESULTS: In the Caucasian kindred, the FH homozygosity can be attributed to two compound heterozygous LDLR damaging variants, an exon 12 p.G592E missense mutation and a novel 3kb exon 1 deletion. By analyzing the 10xG phased data, we ascertained that this deletion allele was most likely to have originated from a Russian ancestor. In the Mexican kindred, the strikingly elevated LDL cholesterol level can be attributed to a homozygous frameshift LDLR variant p.E113fs. CONCLUSIONS: While the application of WES can provide a cost-effective way of identifying the genetic causes of FH, it often lacks sensitivity for detecting structural variants. Our finding of the LDLR exon 1 deletion highlights the broader utility of Linked-Read WGS in detecting SVs in the clinical setting, especially when HoFH patients remain undiagnosed after WES.

Genome maps across 26 human populations reveal population-specific patterns of structural variation.

Large structural variants (SVs) in the human genome are difficult to detect and study by conventional sequencing technologies. With long-range genome analysis platforms, such as optical mapping, one can identify large SVs (>2 kb) across the genome in one experiment. Analyzing optical genome maps of 154 individuals from the 26 populations sequenced in the 1000 Genomes Project, we find that phylogenetic population patterns of large SVs are similar to those of single nucleotide variations in 86% of the human genome, while ~2% of the genome has high structural complexity. We are able to characterize SVs in many intractable regions of the genome, including segmental duplications and subtelomeric, pericentromeric, and acrocentric areas. In addition, we discover ~60 Mb of non-redundant genome content missing in the reference genome sequence assembly. Our results highlight the need for a comprehensive set of alternate haplotypes from different populations to represent SV patterns in the genome.

OMSV enables accurate and comprehensive identification of large structural variations from nanochannel-based single-molecule optical maps.

We present a new method, OMSV, for accurately and comprehensively identifying structural variations (SVs) from optical maps. OMSV detects both homozygous and heterozygous SVs, SVs of various types and sizes, and SVs with or without creating or destroying restriction sites. We show that OMSV has high sensitivity and specificity, with clear performance gains over the latest method. Applying OMSV to a human cell line, we identified hundreds of SVs >2 kbp, with 68 % of them missed by sequencing-based callers. Independent experimental validation confirmed the high accuracy of these SVs. The OMSV software is available at http://yiplab.cse.cuhk.edu.hk/omsv/ .

Bundle Branch Re-Entrant Ventricular Tachycardia: Novel Genetic Mechanisms in a Life-Threatening Arrhythmia.

OBJECTIVES: This study sought to investigate for an underlying genetic etiology in cases of apparent idiopathic bundle branch re-entrant ventricular tachycardia (BBRVT). BACKGROUND: BBRVT is a life-threatening arrhythmia occurring secondary to macro-re-entry within the His-Purkinje system. Although classically associated with dilated cardiomyopathy, BBRVT may also occur in the setting of isolated, unexplained conduction system disease. METHODS: Cases of BBRVT with normal biventricular size and function were recruited from 6 North American centers. Enrollment required a clinically documented wide complex tachycardia and BBRVT proven during invasive electrophysiology study. Study participants were screened for mutations within genes associated with cardiac conduction system disease. Pathogenicity of identified mutations was evaluated using in silico phylogenetic and physicochemical analyses and in vitro biophysical studies. RESULTS: Among 6 cases of idiopathic BBRVT, each presented with hemodynamic compromise and 2 suffered cardiac arrests requiring resuscitation. Putative culprit mutations were identified in 3 of 6 cases, including 2 in SCN5A (Ala1905Gly [novel] and c.4719C>T [splice site mutation]) and 1 in LMNA (Leu327Val [novel]). Biophysical analysis of mutant Ala1905Gly Nav1.5 channels in tsA201 cells revealed significantly reduced peak current density and positive shifts in the voltage-dependence of activation, consistent with a loss-of-function. The SCN5A c.4719C>T splice site mutation has previously been reported as disease-causing in 3 cases of Brugada syndrome, whereas the novel LMNA Leu327Val mutation was associated with a classic laminopathy phenotype. Following catheter ablation, BBRVT was noninducible in all cases and none experienced a clinical recurrence during follow-up. CONCLUSIONS: Our investigation into apparent idiopathic BBRVT has identified the first genetic culprits for this life-threatening arrhythmia, providing further insight into its underlying pathophysiology and emphasizing a potential role for genetic testing in this condition. Our findings also highlight BBRVT as a novel genetic etiology of unexplained sudden cardiac death that can be cured with catheter ablation.

Atrial Fibrillation Associated Genetic Variants and Left Atrial Histology: Evaluation for Molecular Sub-Phenotypes.

INTRODUCTION: Genome wide association studies have identified several single nucleotide polymorphisms (SNPs) associated with atrial fibrillation (AF), but the mechanisms underlying these relationships have not yet been elucidated. Inflammation and fibrosis have been posited as important mechanisms responsible for AF. We sought to investigate the impact of SNP carrier status on inflammation and fibrosis in left atrial appendage tissue. METHODS AND RESULTS: Carrier status of 10 AF-associated SNPs was evaluated on DNA extracted from left atrial appendage tissue in 176 individuals (120 with AF). The presence of inflammation was evaluated through visual quantification of leukocyte infiltration following hematoxylin and eosin staining, while fibrosis was quantified using picrosirius red with fast green staining. Unadjusted and adjusted linear and logistic regression models were utilized to evaluate for an association between SNP carrier status and inflammation and fibrosis. On adjusted logistic regression analysis, the rs7164883 SNP (intronic within HCN4) was associated with a reduced odds of inflammation (odds ratio: 0.42; 95% CI: 0.22-0.81, P = 0.01), and was not associated with fibrosis on adjusted linear regression analysis (ß-coefficient: -0.31; 95% CI: -1.03-0.40, P = 0.40). None of the remaining SNPs exhibited significant associations with left atrial inflammation or fibrosis. CONCLUSIONS: Among 10 AF-associated SNPs, a single genetic variant was associated with reduced left atrial inflammation, while no histologic differences were observed in the remaining 9. The known AF-associated SNPs do not appear to predispose to the development of pro-inflammatory or pro-fibrotic AF sub-phenotypes.

Association of common genetic variation in the protein C pathway genes with clinical outcomes in acute respiratory distress syndrome.

BACKGROUND: Altered plasma levels of protein C, thrombomodulin, and the endothelial protein C receptor are associated with poor clinical outcomes in patients with acute respiratory distress syndrome (ARDS). We hypothesized that common variants in these genes would be associated with mortality as well as ventilator-free and organ failure-free days in patients with ARDS. METHODS: We genotyped linkage disequilibrium-based tag single-nucleotide polymorphisms in the ProteinC, Thrombomodulin and Endothelial Protein C Reptor Genes among 320 self-identified white patients of European ancestry from the ARDS Network Fluid and Catheter Treatment Trial. We then tested their association with mortality as well as ventilator-free and organ-failure free days. RESULTS: The GG genotype of rs1042580 (p = 0.02) and CC genotype of rs3716123 (p = 0.002), both in the thrombomodulin gene, and GC/CC genotypes of rs9574 (p = 0.04) in the endothelial protein C receptor gene were independently associated with increased mortality. An additive effect on mortality (p < 0.001), ventilator-free days (p = 0.01), and organ failure-free days was observed with combinations of these high-risk genotypes. This association was independent of age, severity of illness, presence or absence of sepsis, and treatment allocation. CONCLUSIONS: Genetic variants in thrombomodulin and endothelial protein C receptor genes are additively associated with mortality in ARDS. These findings suggest that genetic differences may be at least partially responsible for the observed associations between dysregulated coagulation and poor outcomes in ARDS.

Impact of bronchopulmonary dysplasia on brain and retina.

Many premature newborns develop bronchopulmonary dysplasia (BPD), a chronic lung disease resulting from prolonged mechanical ventilation and hyperoxia. BPD survivors typically suffer long-term injuries not only to the lungs, but also to the brain and retina. However, currently it is not clear whether the brain and retinal injuries in these newborns are related only to their prematurity, or also to BPD. We investigated whether the hyperoxia known to cause histologic changes in the lungs similar to BPD in an animal model also causes brain and retinal injuries. Sprague Dawley rat pups were exposed to hyperoxia (95% O2, 'BPD' group) or room air (21% O2, 'control' group) from postnatal day 4-14 (P4-14); the rat pups were housed in room air between P14 and P28. At P28, they were sacrificed, and their lungs, brain, and eyes were extracted. Hematoxylin and eosin staining was performed on lung and brain sections; retinas were stained with Toluidine Blue. Hyperoxia exposure resulted in an increased mean linear intercept in the lungs (P<0.0001). This increase was associated with a decrease in some brain structures [especially the whole-brain surface (P=0.02)], as well as a decrease in the thickness of the retinal layers [especially the total retina (P=0.0008)], compared to the room air control group. In addition, a significant negative relationship was observed between the lung structures and the brain (r=-0.49,P=0.02) and retina (r=-0.70,P=0.0008) structures. In conclusion, hyperoxia exposure impaired lung, brain, and retina structures. More severe lung injuries correlated with more severe brain and retinal injuries. This result suggests that the same animal model of chronic neonatal hyperoxia can be used to simultaneously study lung, brain and retinal injuries related to hyperoxia.

Genome-Wide Structural Variation Detection by Genome Mapping on Nanochannel Arrays.

Comprehensive whole-genome structural variation detection is challenging with current approaches. With diploid cells as DNA source and the presence of numerous repetitive elements, short-read DNA sequencing cannot be used to detect structural variation efficiently. In this report, we show that genome mapping with long, fluorescently labeled DNA molecules imaged on nanochannel arrays can be used for whole-genome structural variation detection without sequencing. While whole-genome haplotyping is not achieved, local phasing (across >150-kb regions) is routine, as molecules from the parental chromosomes are examined separately. In one experiment, we generated genome maps from a trio from the 1000 Genomes Project, compared the maps against that derived from the reference human genome, and identified structural variations that are >5 kb in size. We find that these individuals have many more structural variants than those published, including some with the potential of disrupting gene function or regulation.

An internal promoter underlies the difference in disease severity between N- and C-terminal truncation mutations of Titin in zebrafish.

Truncating mutations in the giant sarcomeric protein Titin result in dilated cardiomyopathy and skeletal myopathy. The most severely affected dilated cardiomyopathy patients harbor Titin truncations in the C-terminal two-thirds of the protein, suggesting that mutation position might influence disease mechanism. Using CRISPR/Cas9 technology, we generated six zebrafish lines with Titin truncations in the N-terminal and C-terminal regions. Although all exons were constitutive, C-terminal mutations caused severe myopathy whereas N-terminal mutations demonstrated mild phenotypes. Surprisingly, neither mutation type acted as a dominant negative. Instead, we found a conserved internal promoter at the precise position where divergence in disease severity occurs, with the resulting protein product partially rescuing N-terminal truncations. In addition to its clinical implications, our work may shed light on a long-standing mystery regarding the architecture of the sarcomere.

The bifunctional µ opioid agonist/antioxidant [Dmt(1)]DALDA is a superior analgesic in an animal model of complex regional pain syndrome-type i.

Reactive oxygen species (ROS) play an important role in the development of complex regional pain syndrome-Type I (CRPS-I), as also demonstrated with the chronic post ischemia pain (CPIP) animal model of CRPS-I. We show that morphine and the antioxidant N-acetylcysteine (NAC) act synergistically to reduce mechanical allodynia in CPIP rats. The tetrapeptide amide [Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2) is a potent and selective µ opioid receptor (MOR) agonist with favorable pharmacokinetic properties and with antioxidant activity due to its N-terminal Dmt (2',6'-dimethyltyrosine) residue. In the CPIP model, [Dmt(1)]DALDA was 15-fold more potent than morphine in reversing mechanical allodynia and 4.5-fold more potent as analgesic in the heat algesia test. The results indicate that bifunctional compounds with MOR agonist/antioxidant activity have therapeutic potential for the treatment of CRPS-I.

Targeted deep sequencing reveals no definitive evidence for somatic mosaicism in atrial fibrillation.

BACKGROUND: Studies of ≤15 atrial fibrillation (AF) patients have identified atrial-specific mutations within connexin genes, suggesting that somatic mutations may account for sporadic cases of the arrhythmia. We sought to identify atrial somatic mutations among patients with and without AF using targeted deep next-generation sequencing of 560 genes, including genetic culprits implicated in AF, the Mendelian cardiomyopathies and channelopathies, and all ion channels within the genome. METHODS AND RESULTS: Targeted gene capture and next-generation sequencing were performed on DNA from lymphocytes and left atrial appendages of 34 patients (25 with AF). Twenty AF patients had undergone cardiac surgery exclusively for pulmonary vein isolation and 17 had no structural heart disease. Sequence alignment and variant calling were performed for each atrial-lymphocyte pair using the Burrows-Wheeler Aligner, the Genome Analysis Toolkit, and MuTect packages. Next-generation sequencing yielded a median 265-fold coverage depth (interquartile range, 64-369). Comparison of the 3 million base pairs from each atrial-lymphocyte pair revealed a single potential somatic missense mutation in 3 AF patients and 2 in a single control (12 versus 11%; P=1). All potential discordant variants had low allelic fractions (range, 2.3%-7.3%) and none were detected with conventional sequencing. CONCLUSIONS: Using high-depth next-generation sequencing and state-of-the art somatic mutation calling approaches, no pathogenic atrial somatic mutations could be confirmed among 25 AF patients in a comprehensive cardiac arrhythmia genetic panel. These findings indicate that atrial-specific mutations are rare and that somatic mosaicism is unlikely to exert a prominent role in AF pathogenesis.

Effects of the absence of apolipoprotein e on lipoproteins, neurocognitive function, and retinal function.

IMPORTANCE: The identification of a patient with a rare form of severe dysbetalipoproteinemia allowed the study of the consequences of total absence of apolipoprotein E (apoE). OBJECTIVES: To discover the molecular basis of this rare disorder and to determine the effects of complete absence of apoE on neurocognitive and visual function and on lipoprotein metabolism. DESIGN, SETTING, AND PARTICIPANTS: Whole-exome sequencing was performed on the patient's DNA. He underwent detailed neurological and visual function testing and lipoprotein analysis. Lipoprotein analysis was also performed in the Cardiovascular Research Institute, University of California, San Francisco, on blood samples from the proband's mother, wife, 2 daughters, and normolipidemic control participants. MAIN OUTCOME MEASURES: Whole-exome sequencing, lipoprotein analysis, and neurocognitive function. RESULTS: The patient was homozygous for an ablative APOE frameshift mutation (c.291del, p.E97fs). No other mutations likely to contribute to the phenotype were discovered, with the possible exception of two, in ABCC2 (p.I670T) and LIPC (p.G137R). Despite complete absence of apoE, he had normal vision, exhibited normal cognitive, neurological, and retinal function, had normal findings on brain magnetic resonance imaging, and had normal cerebrospinal fluid levels of ß-amyloid and tau proteins. He had no significant symptoms of cardiovascular disease except a suggestion of myocardial ischemia on treadmill testing and mild atherosclerosis noted on carotid ultrasonography. He had exceptionally high cholesterol content (760 mg/dL; to convert to millimoles per liter, multiply by 0.0259) and a high cholesterol to triglycerides ratio (1.52) in very low-density lipoproteins with elevated levels of small-diameter high-density lipoproteins, including high levels of prebeta-1 high-density lipoprotein. Intermediate-density lipoproteins, low-density lipoproteins, and very low-density lipoproteins contained elevated apoA-I and apoA-IV levels. The patient's apoC-III and apoC-IV levels were decreased in very low-density lipoproteins. Electron microscopy revealed large lamellar particles having electron-opaque cores attached to electron-lucent zones in intermediate-density and low-density lipoproteins. Low-density lipoprotein particle diameters were distributed bimodally. CONCLUSIONS AND RELEVANCE: Despite a profound effect on lipoprotein metabolism, detailed neurocognitive and retinal studies failed to demonstrate any defects. This suggests that functions of apoE in the brain and eye are not essential or that redundant mechanisms exist whereby its role can be fulfilled. Targeted knockdown of apoE in the central nervous system might be a therapeutic modality in neurodegenerative disorders.

Prioritizing causal disease genes using unbiased genomic features.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death in the developed world. Human genetic studies, including genome-wide sequencing and SNP-array approaches, promise to reveal disease genes and mechanisms representing new therapeutic targets. In practice, however, identification of the actual genes contributing to disease pathogenesis has lagged behind identification of associated loci, thus limiting the clinical benefits. RESULTS: To aid in localizing causal genes, we develop a machine learning approach, Objective Prioritization for Enhanced Novelty (OPEN), which quantitatively prioritizes gene-disease associations based on a diverse group of genomic features. This approach uses only unbiased predictive features and thus is not hampered by a preference towards previously well-characterized genes. We demonstrate success in identifying genetic determinants for CVD-related traits, including cholesterol levels, blood pressure, and conduction system and cardiomyopathy phenotypes. Using OPEN, we prioritize genes, including FLNC, for association with increased left ventricular diameter, which is a defining feature of a prevalent cardiovascular disorder, dilated cardiomyopathy or DCM. Using a zebrafish model, we experimentally validate FLNC and identify a novel FLNC splice-site mutation in a patient with severe DCM. CONCLUSION: Our approach stands to assist interpretation of large-scale genetic studies without compromising their fundamentally unbiased nature.

Associations between routine coronary computed tomographic angiography and reduced unnecessary hospital admissions, length of stay, recidivism rates, and invasive coronary angiography in the emergency department triage of chest pain.

OBJECTIVES: This study was designed to assess the effects on resource utilization of routine coronary computed tomographic angiography (CCTA) in triaging chest pain patients in the emergency department (ED). BACKGROUND: The routine use of CCTA for ED evaluation of chest pain is feasible and safe. METHODS: We conducted a retrospective multivariate analysis of data from two risk-matched cohorts of 894 ED patients presenting with chest pain to assess the impact of CCTA versus standard evaluation on admissions rate, length of stay, major adverse cardiovascular event rates, recidivism rates, and downstream resource utilization. RESULTS: The overall admission rate was lower with CCTA (14% vs. 40%; p < 0.001). Standard evaluation was associated with a 5.5-fold greater risk for admission (odds ratio [OR]: 5.53; p < 0.001). Expected ED length of stay with standard evaluation was about 1.6 times longer (OR: 1.55; p < 0.001). There were no differences in the rates of death and acute myocardial infarction within 30 days of the index visit between the two groups. The likelihood of returning to the ED within 30 days for recurrent chest pain was 5 times greater with standard evaluation (OR: 5.06; p = 0.022). Standard evaluation was associated with a 7-fold greater likelihood of invasive coronary angiography without revascularization (OR: 7.17; p < 0.001), while neither group was significantly more likely to receive revascularization (OR: 2.06; p = 0.193). The median radiation dose with CCTA was 5.88 mSv (n = 1039; confidence interval: 5.2 to 6.4). CONCLUSIONS: The routine use of CCTA in ED evaluation of chest pain reduces healthcare resource utilization.

Utilization of coronary computed tomography angiography for exclusion of coronary artery disease in ED patients with low- to intermediate-risk chest pain: a 1-year experience.

OBJECTIVE: We describe our preliminary experience with coronary computed tomography angiography (CCTA) in emergency department (ED) patients with low- to intermediate-risk chest pain. METHODS: A convenience cohort of patients with low- to intermediate-risk acute chest pain presenting to a suburban ED in 2009 were prospectively enrolled if the attending physician ordered a CCTA for possible coronary artery disease. Demographic and clinician data were entered into structured data collection sheets required before any imaging. The results of CCTA were classified as normal, nonobstructive (1%-50% stenosis), and obstructive (>50% stenosis). Outcomes included hospital admission and death within a 6-month follow-up period. RESULTS: In 2009, 507 patients with ED chest pain had a CCTA while in the ED. The median (interquartile range) age was 54 (47-62) years; 51.5% were female. Thrombolysis in myocardial infarction risk scores were 0 (42.6%), 1 (42.2%), 2 (11.8%), 3 (2.4%), and 4 (1.0%). The results of CCTA were normal (n = 363), nonobstructive (n = 123), and obstructive (n = 21). Admission rates by CCTA results were obstructive (90.5%), nonobstructive (4.9%), and normal (3.0%). None of the patients with normal or nonobstructive CCTA died within the 6-month follow-up period (0%; 95% confidence interval, 0-0.9%). CONCLUSIONS: Many ED patients with low- to intermediate-risk chest pain have a normal or nonobstructive CCTA and may be safely discharged from the ED without any associated mortality within the following 6 months.

Mitochondrial DNA sequence variation and risk of pancreatic cancer.

Although the mitochondrial genome exhibits high mutation rates, common mitochondrial DNA (mtDNA) variation has not been consistently associated with pancreatic cancer. Here, we comprehensively examined mitochondrial genomic variation by sequencing the mtDNA of participants (cases = 286, controls = 283) in a San Francisco Bay Area pancreatic cancer case-control study. Five common variants were associated with pancreatic cancer at nominal statistical significance (P < 0.05) with the strongest finding for mt5460g in the ND2 gene [OR = 3.9; 95% confidence interval (CI), 1.5-10; P = 0.004] which encodes an A331T substitution. Haplogroup K was nominally associated with reduced pancreatic cancer risk (OR = 0.32; 95% CI, 0.13-0.76; P = 0.01) when compared with the most common haplogroup, H. A total of 19 haplogroup-specific rare variants yielded nominal statistically significant associations (P < 0.05) with pancreatic cancer risk, with the majority observed in genes involved in oxidative phosphorylation. Weighted-sum statistics were used to identify an aggregate effect of variants in the 22 mitochondrial tRNAs on pancreatic cancer risk (P = 0.02). While the burden of singleton variants in the HV2 and 12S RNA regions was three times higher among European haplogroup N cases than controls, the prevalence of singleton variants in ND4 and ND5 was two to three times higher among African haplogroup L cases than in controls. Together, the results of this study provide evidence that aggregated common and rare variants and the accumulation of singleton variants are important contributors to pancreatic cancer risk.

Association analysis identifies ZNF750 regulatory variants in psoriasis.

BACKGROUND: Mutations in the ZNF750 promoter and coding regions have been previously associated with Mendelian forms of psoriasis and psoriasiform dermatitis. ZNF750 encodes a putative zinc finger transcription factor that is highly expressed in keratinocytes and represents a candidate psoriasis gene. METHODS: We examined whether ZNF750 variants were associated with psoriasis in a large case-control population. We sequenced the promoter and exon regions of ZNF750 in 716 Caucasian psoriasis cases and 397 Caucasian controls. RESULTS: We identified a total of 47 variants, including 38 rare variants of which 35 were novel. Association testing identified two ZNF750 haplotypes associated with psoriasis (p < 0.05). We also identified an excess of rare promoter and 5'untranslated region (UTR) variants in psoriasis cases compared to controls (p = 0.041), whereas there was no significant difference in the number of rare coding and rare 3' UTR variants. Using a promoter functional assay in stimulated human primary keratinocytes, we showed that four ZNF750 promoter and 5' UTR variants displayed a 35-55% reduction of ZNF750 promoter activity, consistent with the promoter activity reduction seen in a Mendelian psoriasis family with a ZNF750 promoter variant. However, the rare promoter and 5' UTR variants identified in this study did not strictly segregate with the psoriasis phenotype within families. CONCLUSIONS: Two haplotypes of ZNF750 and rare 5' regulatory variants of ZNF750 were found to be associated with psoriasis. These rare 5' regulatory variants, though not causal, might serve as a genetic modifier of psoriasis.

A genetic risk score combining ten psoriasis risk loci improves disease prediction.

Psoriasis is a chronic, immune-mediated skin disease affecting 2-3% of Caucasians. Recent genetic association studies have identified multiple psoriasis risk loci; however, most of these loci contribute only modestly to disease risk. In this study, we investigated whether a genetic risk score (GRS) combining multiple loci could improve psoriasis prediction. Two approaches were used: a simple risk alleles count (cGRS) and a weighted (wGRS) approach. Ten psoriasis risk SNPs were genotyped in 2815 case-control samples and 858 family samples. We found that the total number of risk alleles in the cases was significantly higher than in controls, mean 13.16 (SD 1.7) versus 12.09 (SD 1.8), p = 4.577×10(-40). The wGRS captured considerably more risk than any SNP considered alone, with a psoriasis OR for high-low wGRS quartiles of 10.55 (95% CI 7.63-14.57), p = 2.010×10(-65). To compare the discriminatory ability of the GRS models, receiver operating characteristic curves were used to calculate the area under the curve (AUC). The AUC for wGRS was significantly greater than for cGRS (72.0% versus 66.5%, p = 2.13×10(-8)). Additionally, the AUC for HLA-C alone (rs10484554) was equivalent to the AUC for all nine other risk loci combined (66.2% versus 63.8%, p = 0.18), highlighting the dominance of HLA-C as a risk locus. Logistic regression revealed that the wGRS was significantly associated with two subphenotypes of psoriasis, age of onset (p = 4.91×10(-6)) and family history (p = 0.020). Using a liability threshold model, we estimated that the 10 risk loci account for only 11.6% of the genetic variance in psoriasis. In summary, we found that a GRS combining 10 psoriasis risk loci captured significantly more risk than any individual SNP and was associated with early onset of disease and a positive family history. Notably, only a small fraction of psoriasis heritability is captured by the common risk variants identified to date.