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Hepatic nuclear factor-4alpha (HNF-4alpha), a transcription factor involved in the regulation of serum lipid and glucose levels, has recently been associated with type 2 diabetes. The HNF-4alpha gene (HNF4A) resides on chromosome 20q12-q13.1, which, in addition to type 2 diabetes, has also previously been linked to high triglycerides in Finnish familial combined hyperlipidemia (FCHL) families. FCHL, characterized by elevated levels of serum total cholesterol, triglycerides, or both, is a common dyslipidemia observed in up to 20% of patients with premature coronary heart disease. Considering the clear phenotypic overlap between type 2 diabetes and FCHL, both predisposing to high serum triglycerides and glucose intolerance, we tested this gene for association in dyslipidemic families originating from two distinct populations, Finnish and Mexican, and comprising 1,447 subjects. Our data show that common HNF4A variants and haplotypes are associated with elevated serum lipid levels and the metabolic syndrome (P = 0.008-0.04), as well as with elevated glucose parameters (P = 0.008-0.03), using family-based association analysis. Importantly, both Finnish and Mexican families shared two common lipid-associated HNF4A haplotypes (P = 0.005 for total cholesterol and 0.006 for triglycerides). In conclusion, we show for the first time that common HNF4A variants are associated with high serum lipid levels and the metabolic syndrome. [ABSTRACT FROM AUTHOR]

Abstract Background High serum triglyceride (TG) levels is an established risk factor for coronary heart disease (CHD). Fat is stored in the form of TGs in human adipose tissue. We hypothesized that gene co-expression networks in human adipose tissue may be correlated with serum TG levels and help reveal novel genes involved in TG regulation. Methods Gene co-expression networks were constructed from two Finnish and one Mexican study sample using the blockwiseModules R function in Weighted Gene Co-expression Network Analysis (WGCNA). Overlap between TG-associated networks from each of the three study samples were calculated using a Fisher’s Exact test. Gene ontology was used to determine known pathways enriched in each TG-associated network. Results We measured gene expression in adipose samples from two Finnish and one Mexican study sample. In each study sample, we observed a gene co-expression network that was significantly associated with serum TG levels. The TG modules observed in Finns and Mexicans significantly overlapped and shared 34 genes. Seven of the 34 genes (ARHGAP30, CCR1, CXCL16, FERMT3, HCST, RNASET2, SELPG) were identified as the key hub genes of all three TG modules. Furthermore, two of the 34 genes (ARHGAP9, LST1) reside in previous TG GWAS regions, suggesting them as the regional candidates underlying the GWAS signals. Conclusions This study presents a novel adipose gene co-expression network with 34 genes significantly correlated with serum TG across populations.

Background: Genome-wide association studies (GWASs) associate phenotypes and genetic variants across a study cohort. GWASs require large-scale cohorts with both phenotype and genetic sequencing data, limiting studied phenotypes. The Human Phenotype Project is a longitudinal study that has measured a wide range of clinical and biomolecular features from a self-assignment cohort over 5 years. The phenotypes collected are quantitative traits, providing higher-resolution insights into the genetics of complex phenotypes., Methods: We present the results of GWASs and polygenic risk score phenome-wide association studies with 729 clinical phenotypes and 4,043 molecular features from the Human Phenotype Project. This includes clinical traits that have not been previously associated with genetics, including measures from continuous sleep monitoring, continuous glucose monitoring, liver ultrasound, hormonal status, and fundus imaging., Findings: In GWAS of 8,706 individuals, we found significant associations between 169 clinical traits and 1,184 single-nucleotide polymorphisms. We found genes associated with both glycemic control and mental disorders, and we quantify the strength of genetic signals in serum metabolites. In polygenic risk score phenome-wide association studies for clinical traits, we found 16,047 significant associations., Conclusions: The entire set of findings, which we disseminate publicly, provides newfound resolution into the genetic architecture of complex human phenotypes., Funding: E.S. is supported by the Minerva foundation with funding from the Federal German Ministry for Education and Research and by the European Research Council and the Israel Science Foundation., Competing Interests: Declaration of interests H.R., D.W.-V., T.S., and A.D. are employees of Pheno.AI, Ltd, a biomedical data science company from Tel-Aviv, Israel. A.W., A.K., and E.S. are paid consultants to Pheno.AI, Ltd., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Splicing of transcripts is catalyzed by the spliceosome, a mega-complex consisting of hundreds of proteins and five snRNAs, which employs direct interactions. When U1 snRNA forms high-affinity binding, namely more than eight base pairs, with the 5'SS, the result is usually a suppressing effect on the splicing activity. This likely occurs due to the inefficient unwinding of U1/5'SS base-pairing or other regulatory obstructions. Here, we show in vitro and in patient-derived cell lines that pre-microRNAs can modulate the splicing reaction by interacting with U1 snRNA. This leads to reduced binding affinity to the 5'SS, and hence promotes the inclusion of exons containing 5'SS, despite sequence-based high affinity to U1. Application of the mechanism resulted in correction of the splicing defect in the disease-causing VCAN gene from an individual with Wagner syndrome. This pre-miRNA/U1 interaction can regulate the expression of alternatively spliced exons, thus extending the scope of mechanisms regulating splicing., Competing Interests: The authors declare no competing interests., (© 2023.)

Autosomal recessive congenital ichthyosis (ARCI) manifests with generalized scaling often associated with generalized erythema. Mutations in at least 13 different genes have been reported to cause ARCI. Acral peeling skin syndrome (APSS) is a rare autosomal recessive disorder manifesting with peeling over the distal limbs and dorsal surfaces of hands and feet. APSS is mostly due to mutations in TGM5, encoding transglutaminase 5. Both ARCI and APSS are fully penetrant genetic traits. Here, we describe a consanguineous family in which one patient with mild ARCI was found to carry a homozygous mutation in ALOXE3 (c.1238G > A; p.Gly413Asp). The patient was also found to carry a known pathogenic homozygous mutation in TGM5 (c.1335G > C; p.Lys445Asn) but did not display acral peeling skin. Her uncle carried the same homozygous mutation in TGM5 but carried the ALOXE3 mutation in a heterozygous state and showed clinical features typical of APSS. Taken collectively, these observations suggested that the ALOXE3 mutation suppresses the clinical expression of the TGM5 variant. We hypothesized that ALOXE3 deficiency may affect the expression of a protein capable of compensating for the lack of TGM5 expression. Downregulation of ALOXE3 in primary human keratinocytes resulted in increased levels of corneodesmosin, which plays a critical role in the maintenance of cell-cell adhesion in the upper epidermal layers. Accordingly, ectopic corneodesmosin expression rescued the cell-cell adhesion defect caused by TGM5 deficiency in keratinocytes as ascertained by the dispase dissociation assay. The present data thus provide evidence for phenotypic suppression in a human hereditary skin disorder., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: To identify causes of the autosomal-recessive malformation, diencephalic-mesencephalic junction dysplasia (DMJD) syndrome., Methods: Eight families with DMJD were studied by whole-exome or targeted sequencing, with detailed clinical and radiological characterization. Patient-derived induced pluripotent stem cells were derived into neural precursor and endothelial cells to study gene expression., Results: All patients showed biallelic mutations in the nonclustered protocadherin-12 (PCDH12) gene. The characteristic clinical presentation included progressive microcephaly, craniofacial dysmorphism, psychomotor disability, epilepsy, and axial hypotonia with variable appendicular spasticity. Brain imaging showed brainstem malformations and with frequent thinned corpus callosum with punctate brain calcifications, reflecting expression of PCDH12 in neural and endothelial cells. These cells showed lack of PCDH12 expression and impaired neurite outgrowth., Interpretation: DMJD patients have biallelic mutations in PCDH12 and lack of protein expression. These patients present with characteristic microcephaly and abnormalities of white matter tracts. Such pathogenic variants predict a poor outcome as a result of brainstem malformation and evidence of white matter tract defects, and should be added to the phenotypic spectrum associated with PCDH12-related conditions. Ann Neurol 2018;84:646-655., (© 2018 American Neurological Association.)

In breast cancer patients, the lungs are among the first sites of cancer metastasis, and in nearly one quarter of metastatic patients, the exclusive first event. Two common mouse models mimic breast cancer lung colonization and distal metastasis: an orthotopic model and intravenous (IV) cell injections. Gene expression analysis of pulmonary lesions from these two methods demonstrated high inter-model resemblance. However, microRNA (miRNA) expression profiles were not compared. In this study, we compared the overall miRNA expression profiles (miRNome) of the orthotopic and IV breast cancer metastasis models and identified significant miRNome changes between the two models. Overexpression of the most significant candidate, miR-96 or downregulation of its validated gene-target, ABCE1 reduced cancer cells 2D/3D cell movement and proliferation in vitro, and abated tumor growth and metastasis formation in vivo. Human data analysis further strengthened miR-96/ABCE1 role in breast cancer tumor aggression. Taken together, our results indicate that IV- and orthotopic models differ by their miRNome. Specifically in our study, breast cancer aggressiveness was dictated by miR-96 regulating ABCE1. Overall, miRNome analysis of various metastatic cancer models may lead to the identification of candidate genes critical to metastasis development.

Objectives: Although Familial Mediterranean fever (FMF) is categorized as autosomal recessive, frequent exceptions to this model exist and therefore we aimed to search epigenetic modifications in this disease., Methods: Ten M694V homozygous FMF patients (the most severe phenotype) were recruited for this study. Patients with inflammatory flare were excluded. Total RNA was extracted from peripheral blood, and microRNA expression profiled using NanoString nCounter technology. These patients were compared to 10 healthy age- and sex-matched controls., Results: Seven hundred nighty-eight mature human miRNAs were probed, 103 of which had expression levels above the negative control probes. Seven miRNAs showed significant differences in expression in samples from FMF patients compared to healthy controls: four miRNAs were upregulated (miR-144-3p, miR-21-5p, miR-4454, and miR-451a), and three were downregulated (miR-107, let-7d-5p, and miR-148b-3p)., Conclusion: In this pilot study, we identified epigenetic modifications in clinically quiescent FMF patients. More studies are required for exploration of their contribution to FMF pathogenesis and their potential role as clinical biomarkers., Competing Interests: The study was funded by a commercial source: Novartis Inc. The corresponding author hereby declares on behalf of all authors that there are no financial, non-financial professional or personal competing interests between this funder to any of the authors in relationship to this work and manuscript. (Competing interest is defined as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision making, or publication of research or non-research articles submitted to one of the journals). This does not alter our adherence to PLOS ONE policies on sharing data and materials (as detailed online in the guide for authors http://journals.plos.org/plosone/s/competing-interests).

Sequencing large cohorts of ethnically homogeneous individuals yields genetic insights with implications for the entire population rather than a single individual. In order to evaluate the genetic basis of certain diseases encountered at high frequency in the Ashkenazi Jewish population (AJP), as well as to improve variant annotation among the AJP, we examined the entire exome, focusing on specific genes with known clinical implications in 128 Ashkenazi Jews and compared these data to other non-Jewish populations (European, African, South Asian and East Asian). We targeted American College of Medical Genetics incidental finding recommended genes and the Catalogue of Somatic Mutations in Cancer (COSMIC) germline cancer-related genes. We identified previously known disease-causing variants and discovered potentially deleterious variants in known disease-causing genes that are population specific or substantially more prevalent in the AJP, such as in the ATP and HGFAC genes associated with colorectal cancer and pancreatic cancer, respectively. Additionally, we tested the advantage of utilizing the database of the AJP when assigning pathogenicity to rare variants of independent whole-exome sequencing data of 49 Ashkenazi Jew early-onset breast cancer (BC) patients. Importantly, population-based filtering using our AJP database enabled a reduction in the number of potential causal variants in the BC cohort by 36%. Taken together, population-specific sequencing of the AJP offers valuable, clinically applicable information and improves AJP filter annotation.

In this study, we report a family with X-linked recessive syndrome caused by mutated AMMECR1 and characterized by elliptocytosis with or without anemia, midface hypoplasia, proportionate short stature and hearing loss. Recently, mutations in AMMECR1 were reported in two maternal half-brothers, presenting with nephrocalcinosis, midface hypoplasia and, in one of the siblings, deafness and elliptocytosis. AMMECR1 gene is localized in the critical region of contiguous deletion syndrome on Xq22.3 implicated in Alport syndrome, mental retardation, midface hypoplasia, and elliptocytosis (AMME complex). Interestingly, alternative splicing of exon 2, the same exon harboring the truncating mutation, was observed in the proband and in his unaffected mother. Alternative splicing of this exon is predicted to lead to an in-frame deletion. We provide further evidence that mutated AMMECR1 gene is responsible for this clinically recognizable X-linked condition with variable expressivity., (Copyright © 2017. Published by Elsevier B.V.)

Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway., Competing Interests: The authors have declared that no competing interests exist.

Metastasis is the primary cause for mortality in breast cancer. MicroRNAs, gene expression master regulators, constitute an attractive candidate to control metastasis. Here we show that breast cancer metastasis can be prevented by miR-96 or miR-182 treatment, and decipher the mechanism of action. We found that miR-96/miR-182 downregulate Palladin protein levels, thereby reducing breast cancer cell migration and invasion. A common SNP, rs1071738, at the miR-96/miR-182-binding site within the Palladin 3'-UTR abolishes miRNA:mRNA binding, thus diminishing Palladin regulation by these miRNAs. Regulation is successfully restored by applying complimentary miRNAs. A hydrogel-embedded, gold-nanoparticle-based delivery vehicle provides efficient local, selective, and sustained release of miR-96/miR-182, markedly suppressing metastasis in a breast cancer mouse model. Combined delivery of the miRNAs with a chemotherapy drug, cisplatin, enables significant primary tumour shrinkage and metastasis prevention. Our data corroborate the role of miRNAs in metastasis, and suggest miR-96/miR-182 delivery as a potential anti-metastatic drug.

Objectives: Most studies on the genes involved in coronary artery disease (CAD) targeted individuals with angiographically or clinically proven CAD. Focusing on high-risk individuals with normal coronary arteries (NCA) may offer novel insights into the pathogenesis of CAD. We aimed to identify genes putatively protective for development of CAD., Methods: Pooled whole-exome sequencing (WES) was performed on 17 patients with multiple cardiovascular risk factors and NCA and on 17 controls with multivessel CAD. Rare NCA-unique sequence variants were subsequently individually validated using the Fluidigm platform in 100 additional CAD controls and 100 general population controls., Results: In total, 555 100 variants were detected in at least one WES pool in the study group and in none of the control WES pools. For second phase validation, we focused on rare, nonsynonymous variants, resulting in a total of 144 variants in 40 genes, of which 96 were selected for subsequent genotyping. Validation phase genotyping resulted in 19 variants in 16 genes that were found in the NCA group and in none of the CAD controls. The SPTBN5, NID2, and ADAMTSL4 genes harbored sequence variants in more than one CAD-protected patient and none of the 117 CAD controls., Conclusion: Applying WES technology and focusing on individuals seemingly protected from developing CAD successfully identified 19 variants that may offer protection from CAD by undetermined mechanisms. Studying the genetics of high-risk individuals apparently protected from CAD may provide novel insights into the pathogenesis of CAD.

Background: Spiny hyperkeratosis refers to a rare clinical phenotype characterized by nonfollicular keratotic projections and sometimes associated with other acquired and inherited conditions. We describe a case of congenital patterned spiny hyperkeratosis., Methods: To identify the cause of this disorder, we used a combination of whole exome sequencing, direct sequencing and TaqMan assay., Results: We found that the peculiar clinical features displayed by the patient are due to somatic mosaicism for a heterozygous mutation in the GJB2 gene., Conclusion: Because histopathologic examination of two independent biopsies did not reveal porokeratotic eccrine ostial and dermal duct nevus (PEODDN), previously reported to result from somatic mutations in GJB2, it appears that mutations in this gene can cause nevoid spiny hyperkeratosis in the context of PEODDN or as an isolated finding., (© 2016 Wiley Periodicals, Inc.)

Cancer risks in heterozygous mutation carriers of the ATM, BLM, and FANCC genes are controversial. To shed light on this issue, cancer rates were evaluated by cross referencing asymptomatic Israeli heterozygous mutation carriers in the ATM, BLM, and FANCC genes with cancer diagnoses registered at the Israeli National Cancer Registry (INCR). Comparison of observed to expected Standardized Incidence Rates (SIR) was performed. Overall, 474 individuals participated in the study: 378 females; 25 Arab and 31 Jewish ATM carriers, 152 BLM carriers, and 170 FANCC carriers (all Ashkenazim). Age range at genotyping was 19-53 years (mean + SD 30.6 + 5 years). In addition, 96 males were included; 5, 34, and 57 ATM, BLM, and FANCC mutation carriers, respectively. Over 5-16 years from genotyping (4721 person/years), 15 new cancers were diagnosed in mutation carriers: 5 breast, 4 cervical, 3 melanomas, and one each bone sarcoma, pancreatic, and colorectal cancer. No single cancer diagnosis was more prevalent then expected in all groups combined or per gene analyzed. Specifically breast cancer SIR was 0.02-0.77. We conclude that Israeli ATM, BLM, and FANCC heterozygous mutation carriers are not at an increased risk for developing cancer., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Chikungunya virus (CHIKV), an alphavirus and member of the Togaviridae family, is capable of causing severe febrile disease in humans. In December of 2013 the Asian Lineage of CHIKV spread from the Old World to the Americas, spreading rapidly throughout the New World. Given this new emergence in naïve populations we studied the viral genetic diversity present in infected individuals to understand how CHIKV may have evolved during this continuing outbreak., Methodology/principle Findings: We used deep-sequencing technologies coupled with well-established bioinformatics pipelines to characterize the minority variants and diversity present in CHIKV infected individuals from Guadeloupe and Martinique, two islands in the center of the epidemic. We observed changes in the consensus sequence as well as a diverse range of minority variants present at various levels in the population. Furthermore, we found that overall diversity was dramatically reduced after single passages in cell lines. Finally, we constructed an infectious clone from this outbreak and identified a novel 3' untranslated region (UTR) structure, not previously found in nature, that led to increased replication in insect cells., Conclusions/significance: Here we preformed an intrahost quasispecies analysis of the new CHIKV outbreak in the Caribbean. We identified novel variants present in infected individuals, as well as a new 3'UTR structure, suggesting that CHIKV has rapidly evolved in a short period of time once it entered this naïve population. These studies highlight the need to continue viral diversity surveillance over time as this epidemic evolves in order to understand the evolutionary potential of CHIKV.

Whole-exome sequencing (WES) arises as a new approach in diagnosing individuals affected by multigenic and complex phenotypes. Herein, we aim to examine whether WES is useful in screening asymptomatic individuals for actionable interventions, which has not yet been established. Twenty-five healthy adults underwent WES, bioinformatics, and manual curation of their exomes. Six participants (24%) harbored significant, management-changing variants in cancer predisposition genes, American College of Medical Genetics, and genomics reportable cardiac diseases and pharmacogenomic biomarkers that have led to clinical recommendations and interventions. Furthermore, more than 80% of the participants (21) carried 1-3 genetic variants with an associated clinical guideline for an altered drug dosing or administration based on the FDA's table of pharmacogenomics. These results support WES potential not only to answer specific diagnostic questions presented by the relevant personal and/or family history but also to uncover clinically important genetic findings unrelated to the primary indication for sequencing.

Cancer risks and tumor types in male BRCA1 and BRCA2 mutation carriers are still unsettled. Cancer risks in men who were found to harbor a BRCA1 (n = 150) or a BRCA2 (n = 88) mutation or both (n = 2) were assessed by cross referencing with data on cancer occurrence in the Israeli National Cancer Registry. Incidence rates in mutation carriers were compared with men who were counseled, genotyped, and found not to harbor the familial mutation (true negative n = 122), and with standardized incidence rates (SIRs). Of 210 cancer-free individuals at initial counseling, 11 cancers were diagnosed after a mean follow-up of 5.06 ± 4.1 years (1064 person/years) compared with 1/122 in a BRCA true-negative man. The SIR for all BRCA1/2 mutation carriers compared with the rates in the general population were elevated for pancreatic cancer [2.97 (95 % CI 1.83-4.29)] and breast cancer [16.44 (95 % CI 9.65-26.24)]. For prostate cancer these rates were 0.59 (95 % CI 0.4-0.84). Jewish BRCA1/2 mutation carriers are at an increased risk for breast and pancreatic, but not prostate cancer. These cancer risks and the consequent recommendations, if validated, should be transmitted to carriers at test result disclosure.

Background: Low levels of high-density lipoprotein (HDL) cholesterol constitutes a major risk factor for atherosclerosis. Recent studies from our group reported a genetic association between the WW domain-containing oxidoreductase (WWOX) gene and HDL cholesterol levels. Here, through next-generation resequencing, in vivo functional studies and gene microarray analyses, we investigated the role of WWOX in HDL and lipid metabolism., Methods and Results: Using next-generation resequencing of the WWOX region, we first identified 8 variants significantly associated and perfectly segregating with the low-HDL trait in 2 multigenerational French Canadian dyslipidemic families. To understand in vivo functions of WWOX, we used liver-specific Wwox(hep-/-) and total Wwox(-/-) mice models, where we found decreased ApoA-I and Abca1 levels in hepatic tissues. Analyses of lipoprotein profiles in Wwox(-/-), but not Wwox(hep-/-) littermates, also showed marked reductions in serum HDL cholesterol concentrations, concordant with the low-HDL findings observed in families. We next obtained evidence of a sex-specific effect in female Wwox(hep-/-) mice, where microarray analyses revealed an increase in plasma triglycerides and altered lipid metabolic pathways. We further identified a significant reduction in ApoA-I and Lpl and an upregulation in Fas, Angptl4, and Lipg, suggesting that the effects of Wwox involve multiple pathways, including cholesterol homeostasis, ApoA-I/ABCA1 pathway, and fatty acid biosynthesis/triglyceride metabolism., Conclusions: Our data indicate that WWOX disruption alters HDL and lipoprotein metabolism through several mechanisms and may account for the low-HDL phenotype observed in families expressing the WWOX variants. These findings thus describe a novel gene involved in cellular lipid homeostasis, which effects may impact atherosclerotic disease development., (© 2014 American Heart Association, Inc.)

Dyslipidemia and obesity are especially prevalent in populations with Amerindian backgrounds, such as Mexican-Americans, which predispose these populations to cardiovascular disease. Here we design an approach, known as the cross-population allele screen (CPAS), which we conduct prior to a genome-wide association study (GWAS) in 19,273 Europeans and Mexicans, in order to identify Amerindian risk genes in Mexicans. Utilizing CPAS to restrict the GWAS input variants to only those differing in frequency between the two populations, we identify novel Amerindian lipid genes, receptor-related orphan receptor alpha (RORA) and salt-inducible kinase 3 (SIK3), and three loci previously unassociated with dyslipidemia or obesity. We also detect lipoprotein lipase (LPL) and apolipoprotein A5 (APOA5) harbouring specific Amerindian signatures of risk variants and haplotypes. Notably, we observe that SIK3 and one novel lipid locus underwent positive selection in Mexicans. Furthermore, after a high-fat meal, the SIK3 risk variant carriers display high triglyceride levels. These findings suggest that Amerindian-specific genetic architecture leads to a higher incidence of dyslipidemia and obesity in modern Mexicans.

Background: The Mexican population and others with Amerindian heritage exhibit a substantial predisposition to dyslipidemias and coronary heart disease. Yet, these populations remain underinvestigated by genomic studies, and to date, no genome-wide association (GWA) studies have been reported for lipids in these rapidly expanding populations., Methods and Findings: We performed a two-stage GWA study for hypertriglyceridemia and low high-density lipoprotein cholesterol (HDL-C) in Mexicans (n=4361), and identified a novel Mexican-specific genome-wide significant locus for serum triglycerides (TGs) near the Niemann-Pick type C1 protein gene (p=2.43×10(-08)). Furthermore, three European loci for TGs (APOA5, GCKR and LPL), and four loci for HDL-C (ABCA1, CETP, LIPC and LOC55908) reached genome-wide significance in Mexicans. We used cross-ethnic mapping to narrow three European TG GWA loci, APOA5, MLXIPL, and CILP2 that were wide and contained multiple candidate variants in the European scan. At the APOA5 locus, this reduced the most likely susceptibility variants to one, rs964184. Importantly, our functional analysis demonstrated a direct link between rs964184 and postprandial serum apoAV protein levels, supporting rs964184 as the causative variant underlying the European and Mexican GWA signal. Overall, 52 of the 100 reported associations from European lipid GWA meta-analysis generalised to Mexicans. However, in 82 of the 100 European GWA loci, a different variant other than the European lead/best-proxy variant had the strongest regional evidence of association in Mexicans., Conclusions: This first Mexican GWA study of lipids identified a novel GWA locus for high TG levels; used the interpopulation heterogeneity to significantly restrict three previously known European GWA signals, and surveyed whether the European lipid GWA SNPs extend to the Mexican population.

Background: Exome sequencing is a recently implemented method to discover rare mutations for Mendelian disorders. Less is known about its feasibility to identify genes for complex traits. We used exome sequencing to search for rare variants responsible for a complex trait, low levels of serum high-density lipoprotein cholesterol (HDL-C)., Methods and Results: We conducted exome sequencing in a large French-Canadian family with 75 subjects available for study, of which 27 had HDL-C values less than the fifth age-sex-specific population percentile. We captured ≈50 Mb of exonic and transcribed sequences of 3 closely related family members with HDL-C levels less than the fifth age-sex percentile and sequenced the captured DNA. Approximately 82,000 variants were detected in each individual, of which 41 rare nonsynonymous variants were shared by the sequenced affected individuals after filtering steps. Two rare nonsynonymous variants in the ATP-binding cassette, subfamily A (ABC1), member 1 (ABCA1), and lipoprotein lipase genes predicted to be damaging were investigated for cosegregation with the low HDL-C trait in the entire extended family. The carriers of either variant had low HDL-C levels, and the individuals carrying both variants had the lowest HDL-C values. Interestingly, the ABCA1 variant exhibited a sex effect which was first functionally identified, and, subsequently, statistically demonstrated using additional French-Canadian families with ABCA1 mutations., Conclusions: This complex combination of 2 rare variants causing low HDL-C in the extended family would not have been identified using traditional linkage analysis, emphasizing the need for exome sequencing of complex lipid traits in unexplained familial cases.

Objective: Lipoprotein lipase (LPL) is a principal enzyme in lipoprotein metabolism, tissue lipid utilization, and energy metabolism. LPL is synthesized by parenchymal cells in adipose, heart, and muscle tissues followed by secretion to extracellular sites, where lipolyic function is exerted. The catalytic activity of LPL is attained during posttranslational maturation, which involves glycosylation, folding, and subunit assembly within the endoplasmic reticulum. A lipase-chaperone, lipase maturation factor 1 (Lmf1), has recently emerged as a critical factor in this process. Previous studies demonstrated that loss-of-function mutations of Lmf1 result in diminished lipase activity and severe hypertriglyceridemia in mice and human subjects. The objective of this study is to investigate whether, beyond its role as a required factor in lipase maturation, variation in Lmf1 expression is sufficient to modulate LPL activity in vivo., Methods and Results: To assess the effects of Lmf1 overexpression in adipose and muscle tissues, we generated aP2-Lmf1 and Mck-Lmf1 transgenic mice. Characterization of relevant tissues revealed increased LPL activity in both mouse strains. In the omental and subcutaneous adipose depots, Lmf1 overexpression was associated with increased LPL specific activity without changes in LPL mass. In contrast, increased LPL activity was due to elevated LPL protein level in heart and gonadal adipose tissue. To extend these studies to humans, we detected association between LMF1 gene variants and postheparin LPL activity in a dyslipidemic cohort., Conclusions: Our results suggest that variation in Lmf1 expression is a posttranslational determinant of LPL activity.

Atherosclerotic cardiovascular disease (ASCVD) affects more than 1 in 3 American adults. Hypercholesterolemia is a major treatable risk factor for ASCVD, yet many individuals fail to reach target levels of LDL-cholesterol (LDL-C) through the use of statins and lifestyle changes. The E3 ubiquitin ligase myosin regulatory light chain-interacting protein (MYLIP; also known as IDOL) is a recently identified regulator of the LDL receptor (LDLR) pathway. Genome-wide association studies (GWASs) in populations of mixed European descent have identified noncoding variants in the MYLIP region as being associated with LDL-C levels, but no underlying functional variants were pinpointed. In order to fine-map actual susceptibility variants, we studied a population demographically distinct from the discovery population to ensure a different pattern of linkage disequilibrium. Our analysis revealed that in a Mexican population, the nonsynonymous SNP rs9370867, which encodes the N342S amino acid substitution, is an underlying functional variant that was associated with high total cholesterol and accounted for one of the previous significant GWAS signals. Functional characterization showed that the Asn-encoding allele was associated with more potent LDLR degradation and decreased LDL uptake. Mutagenesis of residue 342 failed to affect intrinsic MYLIP E3 ligase activity, but it was critical for LDLR targeting. Our findings suggest that modulation of MYLIP activity can affect LDL-C levels and that pharmacologic inhibition of MYLIP activity might be a useful strategy in the treatment of dyslipidemia and ASCVD.

Objective: Recent genome-wide association studies identified a variant rs7575840 in the apolipoprotein B (APOB) gene region as associated with low-density lipoprotein (LDL) cholesterol. However, the underlying functional mechanism of this variant, which resides 6.5 kb upstream of APOB, has remained unknown. Our objective was to investigate rs7575840 for association with refined apoB-containing lipid particles, for replication in a Mexican population, and for its underlying functional mechanism., Methods and Results: Our data show that rs7575840 is associated with serum apoB levels (P=4.85×10(-10)) and apoB-containing lipid particles, very small very-low-density lipoprotein, intermediate lipoprotein, and LDL particles (P=2×10(-5) to 9×10(-7)) in the Finnish Metabolic Syndrome in Men study sample (n=7710). Fine mapping of the APOB region using 43 single-nucleotide polymorphisms replicated the association of rs7575840 with apoB in a Mexican study sample (n=2666, P=3.33×10(-5)). Furthermore, our transcript analyses of adipose RNA samples from 175 subjects in the Finnish Metabolic Syndrome in Men study indicate that rs7575840 alters expression of APOB (P=1.13×10(-10)) and a regional noncoding RNA (BU630349) (P=7.86×10(-6)) in adipose tissue., Conclusions: It has been difficult to convert genome-wide association study associations into mechanistic insights. Our data show that rs7575840 is associated with serum apoB levels and apoB-containing lipid particles, as well as influencing expression of APOB and a regional transcript BU630349 in adipose tissue. We thus provide evidence how a common genome-wide significant single-nucleotide polymorphism, rs7575840, may affect serum apoB, LDL cholesterol, and total cholesterol levels.

Plasma levels of HDL cholesterol (HDL-C) have a strong inherited basis with heritability estimates of 40-60%. The well-established inverse relationship between plasma HDL-C levels and the risk of coronary artery disease (CAD) has led to an extensive search for genetic factors influencing HDL-C concentrations. Over the past 30 years, candidate gene, genome-wide linkage, and most recently genome-wide association (GWA) studies have identified several genetic variations for plasma HDL-C levels. However, the functional role of several of these variants remains unknown, and they do not always correlate with CAD. In this review, we will first summarize what is known about HDL metabolism, monogenic disorders associated with both low and high HDL-C levels, and candidate gene studies. Then we will focus this review on recent genetic findings from the GWA studies and future strategies to elucidate the remaining substantial proportion of HDL-C heritability. Comprehensive investigation of the genetic factors conferring to low and high HDL-C levels using integrative approaches is important to unravel novel pathways and their relations to CAD, so that more effective means of diagnosis, treatment, and prevention will be identified.

Background and Purpose: Although the Mexican population has a high predisposition to dyslipidemias and premature coronary artery disease, this population is underinvestigated for the genetic factors conferring the high susceptibility. This study attempted to determine these genetic factors., Methods and Results: First, we investigated apolipoprotein B (apoB) levels in Mexican extended families with familial combined hyperlipidemia using a two-step testing strategy. In the screening step, we screened 5721 single-nucleotide polymorphisms (SNPs) for linkage signals with apoB. In the test step, we analyzed the 130 SNPs residing in regions of suggestive linkage signals for association with apoB. We identified significant associations with two SNPs (ie, rs1424032 [P=6.07x10(-6)] and rs1349411 [P=2.72x10(-4)]) that surpassed the significance level for the number of tests performed in the test step (P<3.84x10(-4)). Second, these SNPs were tested for replication in Mexican hyperlipidemic case-control samples. The same risk alleles as in the families with familial combined hyperlipidemia were significantly associated (P<0.05) with apoB in the case-control samples. The rs1349411 resides near the apoB messenger RNA editing enzyme (APOBEC1) involved in the processing of APOB messenger RNA in the small intestine. The rs1424032 resides in a highly conserved noncoding region predicted to function as a regulatory element., Conclusions: We identified two novel variants, rs1349411 and rs1424032, for serum apoB levels in Mexicans.

Background: Although epidemiological studies have demonstrated an increased predisposition to low high-density lipoprotein cholesterol and high triglyceride levels in the Mexican population, Mexicans have not been included in any of the previously reported genome-wide association studies for lipids., Methods and Results: We investigated 6 single-nucleotide polymorphisms associated with triglycerides, 7 with high-density lipoprotein cholesterol, and 1 with both triglycerides and high-density lipoprotein cholesterol in recent Caucasian genome-wide association studies in Mexican familial combined hyperlipidemia families and hypertriglyceridemia case-control study samples. These variants were within or near the genes ABCA1, ANGPTL3, APOA5, APOB, CETP, GALNT2, GCKR, LCAT, LIPC, LPL (2), MMAB-MVK, TRIB1, and XKR6-AMAC1L2. We performed a combined analysis of the family-based and case-control studies (n=2298) using the Z method to combine statistics. Ten of the single-nucleotide polymorphisms were nominally significant and 5 were significant after Bonferroni correction (P=2.20 x 10(-3) to 2.6 x 10(-11)) for the number of tests performed (APOA5, CETP, GCKR, and GALNT2). Interestingly, our strongest signal was obtained for triglycerides with the minor allele of rs964184 (P=2.6 x 10(-11)) in the APOA1/C3/A4/A5 gene cluster region that is significantly more common in Mexicans (27%) than in whites (12%)., Conclusions: It is important to confirm whether known loci have a consistent effect across ethnic groups. We show replication of 5 Caucasian genome-wide association studies lipid associations in Mexicans. The remaining loci will require a comprehensive investigation to exclude or verify their significance in Mexicans. We also demonstrate that rs964184 has a large effect (odds ratio, 1.74) and is more frequent in the Mexican population, and thus it may contribute to the high predisposition to dyslipidemias in Mexicans.

Background: A low level of plasma high-density lipoprotein cholesterol (HDL-C) is a risk factor for cardiovascular disease. HDL particles are modulated by a variety of lipases, including endothelial lipase, a phospholipase present on vascular endothelial cells. The proprotein convertase subtilisin/kexin type 5 (PCSK5) gene product is known to directly inactivate endothelial lipase and indirectly cleave and activate angiopoetin-like protein 3, a natural inhibitor of endothelial lipase. We therefore investigated the effect of human PCSK5 genetic variants on plasma HDL-C levels., Methods and Results: Haplotypes at the PCSK5 locus were examined in 9 multigenerational families that included 60 individuals with HDL-C <10th percentile. Segregation with low HDL-C in 1 family was found. Sequencing of the PCSK5 gene in 12 probands with HDL-C <5th percentile identified 7 novel variants. Using a 2-stage design, we first genotyped these single-nucleotide polymorphisms (SNPs) along with 163 tagSNPs and 12 additional SNPs (n=182 total) in 457 individuals with documented coronary artery disease. We identified 9 SNPs associated with HDL-C (P<0.05), with the strongest results for rs11144782 and rs11144766 (P=0.002 and P=0.005, respectively). The SNP rs11144782 was also associated with very low-density lipoprotein (P=0.039), triglycerides (P=0.049), and total apolipoprotein levels (P=0.022). In stage 2, we replicated the association of rs11144766 with HDL-C (P=0.014) in an independent sample of Finnish low HDL-C families. In a combined analysis of both stages (n=883), region-wide significance of rs11144766 and low HDL-C was observed (unadjusted P=1.86x10(-4) and Bonferroni-adjusted P=0.031)., Conclusions: We conclude that variability at the PCSK5 locus influences HDL-C levels, possibly through the inactivation of endothelial lipase activity, and, consequently, atherosclerotic cardiovascular disease risk.

Objective: Activating transcription factor 6 (ATF6) is a sensor of the endoplasmic reticulum stress response and regulates expression of several key lipogenic genes. We used a 2-stage design to investigate whether ATF6 polymorphisms are associated with lipids in subjects at increased risk for cardiovascular disease (CVD)., Methods and Results: In stage 1, 13 tag-SNPs were tested for association in Dutch samples ascertained for familial combined hyperlipidemia (FCHL) or increased risk for CVD (CVR). In stage 2, we further investigated the SNP with the strongest association from stage 1, a Methionine/Valine substitution at amino-acid 67, in Finnish FCHL families and in subjects with CVR from METSIM, a Finnish population-based cohort. The combined analysis of both stages reached region-wide significance (P=9 x 10(-4)), but this association was not seen in the entire METSIM cohort. Our functional analysis demonstrated that Valine at position 67 augments ATF6 protein and its targets Grp78 and Grp94 as well as increases luciferase expression through Grp78 promoter., Conclusions: A common nonsynonymous variant in ATF6 increases ATF6 protein levels and is associated with cholesterol levels in subjects at increased risk for CVD, but this association was not seen in a population-based cohort. Further replication is needed to confirm the role of this variant in lipids.

Objective: There is increasing physiological evidence in rodents connecting the neuropeptide galanin to triglyceride (TG) levels. We hypothesized that variation in the galanin preproprotein (GAL) gene may contribute to hypertriglyceridemia (HTG) in humans., Methods and Results: We investigated GAL as a TG candidate gene by genotyping 4 tagSNPs in Dutch, Finnish, and Mexican familial combined hyperlipidemia (FCHL) families as well as in white combined hyperlipidemia cases/controls (n=2471). The common allele of rs2187331, residing in the promoter region of GAL, was significantly associated with HTG (probability value=0.00038). In an unascertained population sample of 4463 Finnish males, the rare allele of rs2187331 was associated with higher TGs (probability value=0.0028 to 0.00016). We also observed an allele specific difference with rs2187331 in reporter gene expression and nuclear factor binding in vitro. Furthermore, we detected differential expression of many key lipid genes in adipose tissue based on rs2187331 genotypes., Conclusions: The SNP rs2187331 is associated with HTG in FCHL and white combined hyperlipidemia cases/controls and influences TG levels in the population. Further studies are warranted to elucidate the allelic difference observed between FCHL and the general population. Functional evidence shows that rs2187331 has an allele specific cis-regulatory function and influences the expression of lipid related genes in adipose.

Low serum HDL-cholesterol (HDL-C) is a major risk factor for coronary artery disease. We performed targeted genotyping of a 12.4 Mb linked region on 16q to test for association with low HDL-C by using a regional-tag SNP strategy. We identified one SNP, rs2548861, in the WW-domain-containing oxidoreductase (WWOX) gene with region-wide significance for low HDL-C in dyslipidemic families of Mexican and European descent and in low-HDL-C cases and controls of European descent (p = 6.9 x 10(-7)). We extended our investigation to the population level by using two independent unascertained population-based Finnish cohorts, the cross-sectional METSIM cohort of 4,463 males and the prospective Young Finns cohort of 2,265 subjects. The combined analysis provided p = 4 x 10(-4) to 2 x 10(-5). Importantly, in the prospective cohort, we observed a significant longitudinal association of rs2548861 with HDL-C levels obtained at four different time points over 21 years (p = 0.003), and the T risk allele explained 1.5% of the variance in HDL-C levels. The rs2548861 resides in a highly conserved region in intron 8 of WWOX. Results from our in vitro reporter assay and electrophoretic mobility-shift assay demonstrate that this region functions as a cis-regulatory element whose associated rs2548861 SNP has a specific allelic effect and that the region forms an allele-specific DNA-nuclear-factor complex. In conclusion, analyses of 9,798 subjects show significant association between HDL-C and a WWOX variant with an allele-specific cis-regulatory function.

Objective: Stearoyl-CoA desaturase 1 (SCD1) is the rate-limiting enzyme involved in the synthesis of monounsaturated fatty acids, and in mice SCD1 activity is associated with plasma triglyceride levels. We used the fatty acid desaturation index (the plasma ratio of 18:1/18:0) as a marker of SCD1 activity to investigate the relationship of SCD1 to familial combined hyperlipidemia (FCHL)., Methods and Results: The fatty acid desaturation index was measured in 400 individuals from 18 extended FCHL pedigrees. FCHL-affected individuals exhibited increased SCD1 activity when compared to unrelated controls (P < 0.0001). The fatty acid desaturation index was found to be highly heritable (h(2) = 0.48, P = 2.2 x 10(-11)) in this study sample. QTL analysis in 346 sibling pairs from 18 FCHL families revealed suggestive linkage of the desaturation index to chromosomes 3p26.1 to 3p13 (z = 2.7, P = 0.003), containing the peroxisome proliferator-activated receptor gamma (PPARgamma) gene, and 20p11.21 to 20q13.32 (z = 1.7, P = 0.04), containing the hepatocyte nuclear factor 4, alpha (HNF4alpha) gene. A specific haplotype of HNF4alpha was found to be associated with the desaturation index in these FCHL families (P = 0.002)., Conclusions: Our results demonstrate that the fatty acid desaturation index is a highly heritable trait that is associated with the dyslipidemia observed in FCHL.

Hypertriglyceridemia is a hallmark of many disorders, including metabolic syndrome, diabetes, atherosclerosis and obesity. A well-known cause is the deficiency of lipoprotein lipase (LPL), a key enzyme in plasma triglyceride hydrolysis. Mice carrying the combined lipase deficiency (cld) mutation show severe hypertriglyceridemia owing to a decrease in the activity of LPL and a related enzyme, hepatic lipase (HL), caused by impaired maturation of nascent LPL and hepatic lipase polypeptides in the endoplasmic reticulum (ER). Here we identify the gene containing the cld mutation as Tmem112 and rename it Lmf1 (Lipase maturation factor 1). Lmf1 encodes a transmembrane protein with an evolutionarily conserved domain of unknown function that localizes to the ER. A human subject homozygous for a deleterious mutation in LMF1 also shows combined lipase deficiency with concomitant hypertriglyceridemia and associated disorders. Thus, through its profound effect on lipase activity, LMF1 emerges as an important candidate gene in hypertriglyceridemia.

Objective: Familial combined hyperlipidemia (FCHL) characterized by high serum total cholesterol and/or triglycerides (TGs) is a common dyslipidemia predisposing to coronary artery disease (CAD). Recently, the upstream transcription factor 1 (USF1) was linked and associated with FCHL and TGs in Finnish FCHL families. Here we examined the previously associated rs3737787 SNP in extended Dutch FCHL families (n=532) and in a cohort of US subjects who underwent diagnostic coronary angiography (n=1533)., Methods and Results: In males of the Dutch FCHL families, we observed significant sex-dependent associations between the common allele of rs3737787 and FCHL, TGs, and related metabolic traits (P=0.02 to 0.006). In the U.S. Whites, sex-dependent associations with TGs and related metabolic traits were observed for the common allele of rs3737787 in males (P=0.04 to 0.02) and rare allele in females (P=0.05 to 0.002). This intriguing relationship was further supported by the highly significant genotype x sex interactions observed for TGs in the Dutch and TGs and body mass index (BMI) in U.S. White subjects with CAD (P=0.0005 to 0.00004)., Conclusions: These data show that USF1 influences several cardiovascular risk factors in a sex-dependent manner in Dutch FCHL families and U.S. Whites with CAD. A significant interaction between sex and genotype was shown to affect TGs and BMI.