Differential burden of rare protein truncating variants in Alzheimer's disease patients compared to centenarians.

We compared coding region variants of 53 cognitively healthy centenarians and 45 patients with Alzheimer's disease (AD), all of Ashkenazi Jewish (AJ) ancestry. Despite the small sample size, the known AD risk variant APOE4 reached genome-wide significance, indicating the advantage of utilizing 'super-controls'. We restricted our subsequent analysis to rare variants observed at most once in the 1000 Genomes database and having a minor allele frequency below 2% in our AJ sample. We compared the burden of predicted protein altering variants between cases and controls as normalized by the level of rare synonymous variants. We observed an increased burden among AD subjects for predicted loss-of-function (LoFs) variants defined as stop-gain, frame shift, initiation codon (INIT) and splice site mutations (n = 930, OR = 1.3, P = 1.5×E-5). There was no enrichment across all rare protein altering variants defined as missense plus LoFs, in frame indels and stop-loss variants (n = 13 014, OR = 0.97, P = 0.47). Among LoFs, the strongest burden was observed for INIT (OR = 2.16, P = 0.0097) and premature stop variants predicted to cause non-sense-mediated decay in the majority of transcripts (NMD) (OR = 1.98, P = 0.02). Notably, this increased burden of NMD, INIT and splice variants was more pronounced in a set of 1397 innate immune genes (OR = 4.55, P = 0.0043). Further comparison to additional exomes indicates that the difference in LoF burden originated both from the AD and centenarian sample. In summary, we observed an overall increased burden of rare LoFs in AD subjects as compared to centenarians, and this enrichment is more pronounced for innate immune genes.

High-depth whole genome sequencing of an Ashkenazi Jewish reference panel: enhancing sensitivity, accuracy, and imputation.

While increasingly large reference panels for genome-wide imputation have been recently made available, the degree to which imputation accuracy can be enhanced by population-specific reference panels remains an open question. Here, we sequenced at full-depth (≥ 30×), across two platforms (Illumina X Ten and Complete Genomics, Inc.), a moderately large (n = 738) cohort of samples drawn from the Ashkenazi Jewish population. We developed a series of quality control steps to optimize sensitivity, specificity, and comprehensiveness of variant calls in the reference panel, and then tested the accuracy of imputation against target cohorts drawn from the same population. Quality control (QC) thresholds for the Illumina X Ten platform were identified that permitted highly accurate calling of single nucleotide variants across 94% of the genome. QC procedures also identified numerous regions that are poorly mapped using current reference or alternate assemblies. After stringent QC, the population-specific reference panel produced more accurate and comprehensive imputation results relative to publicly available, large cosmopolitan reference panels, especially in the range of rare variants that may be most critical to further progress in mapping of complex phenotypes. The population-specific reference panel also permitted enhanced filtering of clinically irrelevant variants from personal genomes.

GWAS identifies an NAT2 acetylator status tag single nucleotide polymorphism to be a major locus for skin fluorescence.

AIMS/HYPOTHESIS: Skin fluorescence (SF) is a non-invasive marker of AGEs and is associated with the long-term complications of diabetes. SF increases with age and is also greater among individuals with diabetes. A familial correlation of SF suggests that genetics may play a role. We therefore performed parallel genome-wide association studies of SF in two cohorts. METHODS: Cohort 1 included 1,082 participants, 35-67 years of age with type 1 diabetes. Cohort 2 included 8,721 participants without diabetes, aged 18-90 years. RESULTS: rs1495741 was significantly associated with SF in Cohort 1 (p < 6 × 10(-10)), which is known to tag the NAT2 acetylator phenotype. The fast acetylator genotype was associated with lower SF, explaining up to 15% of the variance. In Cohort 2, the top signal associated with SF (p = 8.3 × 10(-42)) was rs4921914, also in NAT2, 440 bases upstream of rs1495741 (linkage disequilibrium r (2) = 1.0 for rs4921914 with rs1495741). We replicated these results in two additional cohorts, one with and one without type 1 diabetes. Finally, to understand which compounds are contributing to the NAT2-SF signal, we examined 11 compounds assayed from skin biopsies (n = 198): the fast acetylator genotype was associated with lower levels of the AGEs hydroimidazolones of glyoxal (p = 0.017). CONCLUSIONS/INTERPRETATION: We identified a robust association between NAT2 and SF in people with and without diabetes. Our findings provide proof of principle that genetic variation contributes to interindividual SF and that NAT2 acetylation status plays a major role.

Pregnancy complicated by obesity induces global transcript expression alterations in visceral and subcutaneous fat.

Maternal obesity is a significant risk factor for development of both maternal and fetal metabolic complications. Increase in visceral fat and insulin resistance is a metabolic hallmark of pregnancy, yet not much is known how obesity alters adipose cellular function and how this may contribute to pregnancy morbidities. We sought to identify alterations in genome-wide transcription expression in both visceral (omental) and abdominal subcutaneous fat deposits in pregnancy complicated by obesity. Visceral and abdominal subcutaneous fat deposits were collected from normal weight and obese pregnant women (n = 4/group) at the time of scheduled uncomplicated cesarean section. A genome-wide expression array (Affymetrix Human Exon 1.0 st platform), validated by quantitative real-time PCR, was utilized to establish the gene transcript expression profile in both visceral and abdominal subcutaneous fat in normal weight and obese pregnant women. Global alteration in gene expression was identified in pregnancy complicated by obesity. These regions of variations led to identification of indolethylamine N-methyltransferase, tissue factor pathway inhibitor-2, and ephrin type-B receptor 6, not previously associated with fat metabolism during pregnancy. In addition, subcutaneous fat of obese pregnant women demonstrated increased coding protein transcripts associated with apoptosis as compared to lean counterparts. Global alteration of gene expression in adipose tissue may contribute to adverse pregnancy outcomes associated with obesity.

Novel candidate genes putatively involved in stress fracture predisposition detected by whole-exome sequencing.

While genetic factors in all likelihood contribute to stress fracture (SF) pathogenesis, a few studies focusing on candidate genes have previously been reported. The objective of this study is to gain better understanding on the genetic basis of SF in a gene-naive manner. Exome sequence capture followed by massive parallel sequencing of two pooled DNA samples from Israeli combat soldiers was employed: cases with high grade SF and ethnically matched healthy controls. The resulting sequence variants were individually verified using the Sequenom™ platform and the contribution of the genetic alterations was validated in a second cohort of cases and controls. In the discovery set that included DNA pool of cases (n = 34) and controls (n = 60), a total of 1174 variants with >600 reads/variant/DNA pool were identified, and 146 (in 127 genes) of these exhibited statistically significant (P < 0·05) different rates between SF cases and controls after multiple comparisons correction. Subsequent validation of these 146 sequence variants individually in a total of 136 SF cases and 127 controls using the Sequenom™ platform validated 20/146 variants. Of these, three missense mutations (rs7426114, rs4073918, rs3752135 in the NEB, SLC6A18 and SIGLEC12 genes, respectively) and three synonymous mutations (rs2071856, rs2515941, rs716745 in the ELFN2, GRK4, LRRC55 genes) displayed significant different rates in SF cases compared with controls. Exome sequencing seemingly unravelled novel candidate genes as involved in SF pathogenesis and predisposition.

Genome-wide SNP scan of pooled DNA reveals nonsense mutation in FGF20 in the scaleless line of featherless chickens.

BACKGROUND: Scaleless (sc/sc) chickens carry a single recessive mutation that causes a lack of almost all body feathers, as well as foot scales and spurs, due to a failure of skin patterning during embryogenesis. This spontaneous mutant line, first described in the 1950s, has been used extensively to explore the tissue interactions involved in ectodermal appendage formation in embryonic skin. Moreover, the trait is potentially useful in tropical agriculture due to the ability of featherless chickens to tolerate heat, which is at present a major constraint to efficient poultry meat production in hot climates. In the interests of enhancing our understanding of feather placode development, and to provide the poultry industry with a strategy to breed heat-tolerant meat-type chickens (broilers), we mapped and identified the sc mutation. RESULTS: Through a cost-effective and labour-efficient SNP array mapping approach using DNA from sc/sc and sc/+ blood sample pools, we map the sc trait to chromosome 4 and show that a nonsense mutation in FGF20 is completely associated with the sc/sc phenotype. This mutation, common to all sc/sc individuals and absent from wild type, is predicted to lead to loss of a highly conserved region of the FGF20 protein important for FGF signalling. In situ hybridisation and quantitative RT-PCR studies reveal that FGF20 is epidermally expressed during the early stages of feather placode patterning. In addition, we describe a dCAPS genotyping assay based on the mutation, developed to facilitate discrimination between wild type and sc alleles. CONCLUSIONS: This work represents the first loss of function genetic evidence supporting a role for FGF ligand signalling in feather development, and suggests FGF20 as a novel central player in the development of vertebrate skin appendages, including hair follicles and exocrine glands. In addition, this is to our knowledge the first report describing the use of the chicken SNP array to map genes based on genotyping of DNA samples from pooled whole blood. The identification of the sc mutation has important implications for the future breeding of this potentially useful trait for the poultry industry, and our genotyping assay can facilitate its rapid introgression into production lines.

The ubiquitin E3 ligase POSH regulates calcium homeostasis through spatial control of Herp.

The ubiquitin (Ub) domain protein Herp plays a crucial role in the maintenance of calcium homeostasis during endoplasmic reticulum (ER) stress. We now show that Herp is a substrate as well as an activator of the E3 Ub ligase POSH. Herp-mediated POSH activation requires the Ubl domain and exclusively promotes lysine-63-linked polyubiquitination. Confocal microscopy demonstrates that Herp resides mostly in the trans-Golgi network, but, shortly after calcium perturbation by thapsigargin (Tpg), it appears mainly in the ER. Substitution of all lysine residues within the Ubl domain abolishes lysine-63-linked polyubiquitination of Herp in vitro and calcium-induced Herp relocalization that is also abrogated by the overexpression of a dominant-negative POSHV14A. A correlation exists between the kinetics of Tpg-induced Herp relocalization and POSH-dependent polyubiquitination. Finally, the overexpression of POSH attenuates, whereas the inhibition of POSH by the expression of POSHV14A or by RNA interference enhances Tpg-induced calcium burst. Altogether, these results establish a critical role for POSH-mediated ubiquitination in the maintenance of calcium homeostasis through the spatial control of Herp.

Novel ultra-rare exonic variants identified in a founder population implicate cadherins in schizophrenia.

The identification of rare variants associated with schizophrenia has proven challenging due to genetic heterogeneity, which is reduced in founder populations. In samples from the Ashkenazi Jewish population, we report that schizophrenia cases had a greater frequency of novel missense or loss of function (MisLoF) ultra-rare variants (URVs) compared to controls, and the MisLoF URV burden was inversely correlated with polygenic risk scores in cases. Characterizing 141 "case-only" genes (MisLoF URVs in ≥3 cases with none in controls), the cadherin gene set was associated with schizophrenia. We report a recurrent case mutation in PCDHA3 that results in the formation of cytoplasmic aggregates and failure to engage in homophilic interactions on the plasma membrane in cultured cells. Modeling purifying selection, we demonstrate that deleterious URVs are greatly overrepresented in the Ashkenazi population, yielding enhanced power for association studies. Identification of the cadherin/protocadherin family as risk genes helps specify the synaptic abnormalities central to schizophrenia.

PRMT1 inhibition induces differentiation of colon cancer cells.

Differentiation therapy has been recently revisited as a prospective approach in cancer therapy by targeting the aberrant growth, and repairing the differentiation and cell death programs of cancer cells. However, differentiation therapy of solid tumors is a challenging issue and progress in this field is limited. We performed High Throughput Screening (HTS) using a novel dual multiplex assay to discover compounds, which induce differentiation of human colon cancer cells. Here we show that the protein arginine methyl transferase (PRMT) type 1 inhibitor, MS023, is a potent inducer of colon cancer cell differentiation with a large therapeutic window. Differentiation changes in the highly aggressive human colon cancer cell line (HT-29) were proved by proteomic and genomic approaches. Growth of HT-29 xenograft in nude mice was significantly delayed upon MS023 treatment and immunohistochemistry of tumor indicated differentiation changes. These findings may lead to development of clinically effective anti-cancer drugs based on the mechanism of cancer cell differentiation.

Four Genome-Wide Association Studies Identify New Extreme Longevity Variants.

The search for the genetic determinants of extreme human longevity has been challenged by the phenotype's rarity and its nonspecific definition by investigators. To address these issues, we established a consortium of four studies of extreme longevity that contributed 2,070 individuals who survived to the oldest one percentile of survival for the 1900 U.S. birth year cohort. We conducted various analyses to discover longevity-associated variants (LAV) and characterized those LAVs that differentiate survival to extreme age at death (eSAVs) from those LAVs that become more frequent in centenarians because of mortality selection (eg, survival to younger years). The analyses identified new rare variants in chromosomes 4 and 7 associated with extreme survival and with reduced risk for cardiovascular disease and Alzheimer's disease. The results confirm the importance of studying truly rare survival to discover those combinations of common and rare variants associated with extreme longevity and longer health span.

The GH receptor exon 3 deletion is a marker of male-specific exceptional longevity associated with increased GH sensitivity and taller stature.

Although both growth hormone (GH) and insulin-like growth factor 1 (IGF-1) signaling were shown to regulate life span in lower organisms, the role of GH signaling in human longevity remains unclear. Because a GH receptor exon 3 deletion (d3-GHR) appears to modulate GH sensitivity in humans, we hypothesized that this polymorphism could play a role in human longevity. We report a linear increased prevalence of d3-GHR homozygosity with age in four independent cohorts of long-lived individuals: 841 participants [567 of the Longevity Genes Project (LGP) (8% increase; P = 0.01), 152 of the Old Order Amish (16% increase; P = 0.02), 61 of the Cardiovascular Health Study (14.2% increase; P = 0.14), and 61 of the French Long-Lived Study (23.5% increase; P = 0.02)]. In addition, mega analysis of males in all cohorts resulted in a significant positive trend with age (26% increase; P = 0.007), suggesting sexual dimorphism for GH action in longevity. Further, on average, LGP d3/d3 homozygotes were 1 inch taller than the wild-type (WT) allele carriers (P = 0.05) and also showed lower serum IGF-1 levels (P = 0.003). Multivariate regression analysis indicated that the presence of d3/d3 genotype adds approximately 10 years to life span. The LGP d3/d3-GHR transformed lymphocytes exhibited superior growth and extracellular signal-regulated kinase activation, to GH treatment relative to WT GHR lymphocytes (P < 0.01), indicating a GH dose response. The d3-GHR variant is a common genetic polymorphism that modulates GH responsiveness throughout the life span and positively affects male longevity.

New Locus for Skin Intrinsic Fluorescence in Type 1 Diabetes Also Associated With Blood and Skin Glycated Proteins.

Skin fluorescence (SF) noninvasively measures advanced glycation end products (AGEs) in the skin and is a risk indicator for diabetes complications. N-acetyltransferase 2 (NAT2) is the only known locus influencing SF. We aimed to identify additional genetic loci influencing SF in type 1 diabetes (T1D) through a meta-analysis of genome-wide association studies (N = 1,359) including Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) and Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR). A locus on chromosome 1, rs7533564 (P = 1.9 × 10(-9)), was associated with skin intrinsic fluorescence measured by SCOUT DS (excitation 375 nm, emission 435-655 nm), which remained significant after adjustment for time-weighted HbA1c (P = 1.7 × 10(-8)). rs7533564 was associated with mean HbA1c in meta-analysis (P = 0.0225), mean glycated albumin (P = 0.0029), and glyoxal hydroimidazolones (P = 0.049), an AGE measured in skin biopsy collagen, in DCCT. rs7533564 was not associated with diabetes complications in DCCT/EDIC or with SF in subjects without diabetes (nondiabetic [ND]) (N = 8,721). In conclusion, we identified a new locus associated with SF in T1D subjects that did not show similar effect in ND subjects, suggesting a diabetes-specific effect. This association needs to be investigated in type 2 diabetes.

Genome-wide analysis identifies 12 loci influencing human reproductive behavior.

The genetic architecture of human reproductive behavior-age at first birth (AFB) and number of children ever born (NEB)-has a strong relationship with fitness, human development, infertility and risk of neuropsychiatric disorders. However, very few genetic loci have been identified, and the underlying mechanisms of AFB and NEB are poorly understood. We report a large genome-wide association study of both sexes including 251,151 individuals for AFB and 343,072 individuals for NEB. We identified 12 independent loci that are significantly associated with AFB and/or NEB in a SNP-based genome-wide association study and 4 additional loci associated in a gene-based effort. These loci harbor genes that are likely to have a role, either directly or by affecting non-local gene expression, in human reproduction and infertility, thereby increasing understanding of these complex traits.

Disease variants in genomes of 44 centenarians.

To identify previously reported disease mutations that are compatible with extraordinary longevity, we screened the coding regions of the genomes of 44 Ashkenazi Jewish centenarians. Individual genome sequences were generated with 30× coverage on the Illumina HiSeq 2000 and single-nucleotide variants were called with the genome analysis toolkit (GATK). We identified 130 coding variants that were annotated as "pathogenic" or "likely pathogenic" based on the ClinVar database and that are infrequent in the general population. These variants were previously reported to cause a wide range of degenerative, neoplastic, and cardiac diseases with autosomal dominant, autosomal recessive, and X-linked inheritance. Several of these variants are located in genes that harbor actionable incidental findings, according to the recommendations of the American College of Medical Genetics. In addition, we found risk variants for late-onset neurodegenerative diseases, such as the APOE ε4 allele that was even present in a homozygous state in one centenarian who did not develop Alzheimer's disease. Our data demonstrate that the incidental finding of certain reported disease variants in an individual genome may not preclude an extraordinarily long life. When the observed variants are encountered in the context of clinical sequencing, it is thus important to exercise caution in justifying clinical decisions.

Epigenetic genome-wide association methylation in aging and longevity.

The aging phenotype is the result of a complex interaction between genetic, epigenetic and environmental factors. Evidence suggests that epigenetic changes (i.e., a set of reversible, heritable changes in gene function or other cell phenotype that occurs without a change in DNA sequence) may affect the aging process and may be one of the central mechanisms by which aging predisposes to many age-related diseases. The total number of altered methylation sites increases with increasing age, such that they could serve as marker for chronological age. This article systematically highlights the advances made in the field of epigenomics and their contribution to the understanding of the complex physiology of aging, lifespan and age-associated diseases.

The E3 ubiquitin-ligase Bmi1/Ring1A controls the proteasomal degradation of Top2alpha cleavage complex - a potentially new drug target.

BACKGROUND: The topoisomerases Top1, Top2alpha and Top2beta are important molecular targets for antitumor drugs, which specifically poison Top1 or Top2 isomers. While it was previously demonstrated that poisoned Top1 and Top2beta are subject to proteasomal degradation, this phenomena was not demonstrated for Top2alpha. METHODOLOGY/PRINCIPAL FINDINGS: We show here that Top2alpha is subject to drug induced proteasomal degradation as well, although at a lower rate than Top2beta. Using an siRNA screen we identified Bmi1 and Ring1A as subunits of an E3 ubiquitin ligase involved in this process. We show that silencing of Bmi1 inhibits drug-induced Top2alpha degradation, increases the persistence of Top2alpha-DNA cleavage complex, and increases Top2 drug efficacy. The Bmi1/Ring1A ligase ubiquitinates Top2alpha in-vitro and cellular overexpression of Bmi1 increases drug induced Top2alpha ubiquitination. A small-molecular weight compound, identified in a screen for inhibitors of Bmi1/Ring1A ubiquitination activity, also prevents Top2alpha ubiquitination and drug-induced Top2alpha degradation. This ubiquitination inhibitor increases the efficacy of topoisomerase 2 poisons in a synergistic manner. CONCLUSIONS/SIGNIFICANCE: The discovery that poisoned Top2alpha is undergoing proteasomal degradation combined with the involvement of Bmi1/Ring1A, allowed us to identify a small molecule that inhibits the degradation process. The Bmi1/Ring1A inhibitor sensitizes cells to Top2 drugs, suggesting that this type of drug combination will have a beneficial therapeutic outcome. As Bmi1 is also a known oncogene, elevated in numerous types of cancer, the identified Bmi1/Ring1A ubiquitin ligase inhibitors can also be potentially used to directly target the oncogenic properties of Bmi1.

The trans-Golgi network-associated human ubiquitin-protein ligase POSH is essential for HIV type 1 production.

HIV type 1 (HIV-1) was shown to assemble either at the plasma membrane or in the membrane of late endosomes. Now, we report an essential role for human ubiquitin ligase POSH (Plenty of SH3s; hPOSH), a trans-Golgi network-associated protein, in the targeting of HIV-1 to the plasma membrane. Small inhibitory RNA-mediated silencing of hPOSH ablates virus secretion and Gag plasma membrane localization. Reintroduction of native, but not a RING finger mutant, hPOSH restores virus release and Gag plasma membrane localization in hPOSH-depleted cells. Furthermore, expression of the RING finger mutant hPOSH inhibits virus release and induces accumulation of intracellular Gag in normal cells. Together, our results identify a previously undescribed step in HIV biogenesis and suggest a direct function for hPOSH-mediated ubiquitination in protein sorting at the trans-Golgi network. Consequently, hPOSH may be a useful host target for therapeutic intervention.