Complete genome sequences of three Prunus-infecting nepoviruses: apricot latent ringspot virus, myrobalan latent ringspot virus, and a novel virus from smooth stone peach (Prunus mira Koehne).

The complete genome sequences of two poorly studied Prunus-infecting nepoviruses, apricot latent ringspot virus (ALRSV) and myrobalan latent ringspot virus (MLRSV) were determined, confirming that they are members of subgroup C. Serological, biological, and molecular data, in particular a low level (58.8%) of amino acid sequence identity in the coat protein, suggest that ALRSV and MLRSV should be considered taxonomically distinct. In addition, data mining of public RNASeq data from wild and ornamental Prunus identified two contigs representing the nearly complete genome of a new subgroup A nepovirus from a smooth stone peach (Prunus mira) dataset (SRR8369794) from the Himalayas, for which the name "Prunus mira virus A" is proposed.

Establishing the relationship between familial dysbetalipoproteinemia and genetic variants in the APOE gene.

Familial Dysbetalipoproteinemia (FD) is the second most common monogenic dyslipidemia and is associated with a very high cardiovascular risk due to cholesterol-enriched remnant lipoproteins. FD is usually caused by a recessively inherited variant in the APOE gene (ε2ε2), but variants with dominant inheritance have also been described. The typical dysbetalipoproteinemia phenotype has a delayed onset and requires a metabolic hit. Therefore, the diagnosis of FD should be made by demonstrating both the genotype and dysbetalipoproteinemia phenotype. Next Generation Sequencing is becoming more widely available and can reveal variants in the APOE gene for which the relation with FD is unknown or uncertain. In this article, two approaches are presented to ascertain the relationship of a new variant in the APOE gene with FD. The comprehensive approach consists of determining the pathogenicity of the variant and its causal relationship with FD by confirming a dysbetalipoproteinemia phenotype, and performing in vitro functional tests and, optionally, in vivo postprandial clearance studies. When this is not feasible, a second, pragmatic approach within reach of clinical practice can be followed for individual patients to make decisions on treatment, follow-up, and family counseling.

Genetic and Mechanistic Insights into the Modulation of Circulating Lipoprotein (a) Concentration by Apolipoprotein E Isoforms.

PURPOSE OF REVIEW: Lipoprotein (a) [Lp(a)] is a highly atherogenic lipoprotein species. A unique feature of Lp(a) is the strong genetic determination of its concentration. The LPA gene is responsible for up to 90% of the variance in Lp(a), but other genes also have an impact. RECENT FINDINGS: Genome-wide associations studies indicate that the APOE gene, encoding apolipoprotein E (apoE), is the second most important locus modulating Lp(a) concentrations. Population studies clearly show that carriers of the apoE2 variant (ε2) display reduced Lp(a) levels, the lowest concentrations being observed in ε2/ε2 homozygotes. This genotype can lead predisposed adults to develop dysbetalipoproteinemia, a lipid disorder characterized by sharp elevations in cholesterol and triglycerides. However, dysbetalipoproteinemia does not significantly modulate circulating Lp(a). Mechanistically, apoE appears to impair the production but not the catabolism of Lp(a). These observations underline the complexity of Lp(a) metabolism and provide key insights into the pathways governing Lp(a) synthesis and secretion.

Novel PCSK9 (Proprotein Convertase Subtilisin Kexin Type 9) Variants in Patients With Familial Hypercholesterolemia From Cape Town.

OBJECTIVE: Familial hypercholesterolemia (FH) is characterized by elevated low-density lipoprotein-cholesterol and markedly increased cardiovascular risk. In patients with a genetic diagnosis, low-density lipoprotein receptor (LDLR) mutations account for >90% of cases, apolipoprotein B (APOB) mutations for ≈5% of cases, while proprotein convertase subtilisin kexin type 9 (PCSK9) gain of function mutations are rare (<1% of cases). We aimed to evaluate the functional impact of several novel PCSK9 variants in a cohort of patients with FH by genetic cascade screening and in vitro functionality assays. Approach and Results: Patients with clinically diagnosed FH underwent genetic analysis of LDLR, and if negative, sequential testing of APOB and PCSK9. We analyzed cosegregation of hypercholesterolemia with novel PCSK9 variants. Gain of function status was determined by in silico analyses and validated by in vitro functionality assays. Among 1055 persons with clinical FH, we identified nonsynonymous PCSK9 variants in 27 (2.6%) patients and 7 of these carried one of the 4 previously reported gain of function variants. In the remaining 20 patients with FH, we identified 7 novel PCSK9 variants. The G516V variant (c.1547G>T) was found in 5 index patients and cascade screening identified 15 additional carriers. Low-density lipoprotein-cholesterol levels were higher in these 15 carriers compared with the 27 noncarriers (236±73 versus 124±35 mg/dL; P<0.001). In vitro studies demonstrated the pathogenicity of the G516V variant. CONCLUSIONS: In our study, 1.14% of cases with clinical FH were clearly attributable to pathogenic variants in PCSK9. Pathogenicity is established beyond doubt for the G516V variant.

Identification of two novel putative satellite RNAs with hammerhead structures in the virome of French and Spanish carrot samples.

Carrot virome analysis using high-throughput sequencing revealed the presence of two RNA molecules with properties of satellite RNAs that are homologous to the satellite RNA of cereal yellow dwarf virus-RPV (CYDV-RPV). Satellite 1 is 298 nt long, while satellite 2 is 368 nt long. Their positive and negative genome strands contain hammerhead ribozymes similar to those found in other self-cleaving satellite RNAs. While both satellites were detected in Spanish carrot populations, only satellite 2 was found in French carrot populations. The most likely helper virus for these two satellites is carrot red leaf virus (CtRLV), which, like CYDV-RPV, is a polerovirus.

Cerebrotendinous xanthomatosis without neurological involvement.

BACKGROUND: Cerebrotendinous xanthomatosis (CTX) is an autosomal recessively inherited inborn error of metabolism. Neurological symptoms are considered to be a clinical hallmark of untreated adult patients. We describe a 'milder CTX phenotype', without neurological involvement. METHODS: We performed a retrospective patient file study in 79 genetically confirmed Dutch patients with CTX (55 patients aged ≥ 21 years) to study the clinical heterogeneity of CTX. We studied the frequency of adult patients with CTX without neurological involvement at diagnosis, in our Dutch cohort, and included a family from South Africa and patients from Italy, USA, Chile and Asia from the literature. RESULTS: In total, we describe 19 adult patients with CTX from 16 independent families, without neurological symptoms at diagnosis. A relatively small percentage (21%, n = 4) had a history of cataract. The majority, 84% (n = 16), presented with tendon xanthomas as the sole or predominant feature. The majority of patients showed increased plasma cholesterol levels. No correlation was found between this 'milder phenotype', the cholestanol levels and the CYP27A1 genotype. In addition, we describe three novel mutations in the CYP27A1 gene. CONCLUSIONS: This study shows the clinical heterogeneity of CTX, highlighting the existence of a 'milder phenotype', that is without neurological involvement at diagnosis. Adult patients with CTX may present with tendon xanthomas as the sole or predominant feature, mimicking familial hypercholesterolemia. It is important to realize that the absence of neurological symptoms does not rule out the development of future neurological symptoms. As CTX is a treatable disorder, early diagnosis and initiation of treatment when additional clinical signs occur is therefore essential.

Apolipoprotein E and Atherosclerosis.

PURPOSE OF REVIEW: The functions, genetic variations and impact of apolipoprotein E on lipoprotein metabolism in general are placed in the context of clinical practice dealing with moderate dyslipidaemia as well as dysbetalipoproteinemia, a highly atherogenic disorder and lipoprotein glomerulopathy. RECENT FINDINGS: Additional variants of apolipoprotein E and participation of apolipoprotein E in inflammation are of interest. The mostly favourable effects of apolipoprotein E2 as well as the atherogenic nature of apolipoproteinE4, which has an association with cognitive impairment, are confirmed. The contribution of remnant lipoproteins of triglyceride-rich lipoproteins, of which dysbetalipoproteinemia represents an extreme, is explored in atherosclerosis. Mimetic peptides may present new therapeutic approaches. Apolipoprotein E is an important determinant of the lipid profile and cardiovascular health in the population at large and can precipitate dysbetalipoproteinemia and glomerulopathy. Awareness of apolipoprotein E polymorphisms should improve medical care.

Complete genome sequence of a novel grapevine-infecting member of the genus Polerovirus, grapevine polerovirus 1.

Double-stranded RNAs and total RNAs purified from grapevine (Vitis vinifera) phloem scrapings of two varieties held in the INRAE (France) grapevine germplasm collection were analyzed by high-throughput sequencing. BLAST annotation revealed contigs with homology to Polerovirus genus members. The full genome sequence of one isolate (KT) was determined (5651 nucleotides [nt]), and a partial sequence representing about half of the genome was assembled for a second isolate (KS) that was found to share 95% nt sequence identity with the KT isolate. The genome has a typical polerovirus organization, containing six open reading frames (ORFs) as well as a putative additional ORF3a. Based on genome organization and phylogenetic relationships, the new virus belongs to the genus Polerovirus but, similar to the recently described persimmon polerovirus 1, is characterized by a highly divergent coat-protein/readthrough domain. Considering the species demarcation criteria for the family Luteoviridae, these two isolates, together with a closely related sequence recently deposited in the GenBank database (LC507098), represent a new Polerovirus species for which the name "Grapevine polerovirus 1" is proposed.

Complete genome sequence of cherry virus T, a novel cherry-infecting tepovirus.

Double-stranded RNA and total RNA purified from sour cherry leaves (Prunus cerasus, cv. Amarelka Chvalkovicka) was analyzed by high-throughput sequencing. BLAST annotation identified contigs with homology to several already known cherry-infecting viruses (prune dwarf virus, prunus necrotic ringspot virus, prunus virus F, little cherry virus 1) as well as contigs with sequences more distantly related to those of members of the family Betaflexiviridae and in particular to prunus virus T of the genus Tepovirus. The full genome sequence of a putative virus (6,847 nucleotides [nt]; GenBank no. MT090966) was assembled and completed at the genome ends. The genome has a typical tepovirus organization, containing three overlapping open reading frames (ORFs), encoding a replication-associated protein, a movement protein and a capsid protein, respectively. Both its genome organization and its phylogenetic relationships show that the virus belongs to the genus Tepovirus, but considering the species demarcation criteria for the family Betaflexiviridae, it appears to represent a novel virus species, and we propose the name "cherry virus T" (ChVT) for this virus.

Lomitapide and Mipomersen-Inhibiting Microsomal Triglyceride Transfer Protein (MTP) and apoB100 Synthesis.

PURPOSE OF REVIEW: The goal of this review is to evaluate the role of inhibiting the synthesis of lipoproteins when there is no or little residual LDL-receptor function as in patients with homozygous familial hypercholesterolaemia. Lomitapide is administered orally once a day while mipomersen is given by subcutaneous injection once a week. Lomitapide inhibits microsomal triglyceride transfer protein while mipomersen is an antisense oligonucleotide directed against apoB100. RECENT FINDINGS: The pivotal registration trials for lomitapide and mipomersen were published in 2013 and 2010, respectively. More recently published data from extension trials and cohort studies provides additional information on long-term safety and efficacy. The mean LDL cholesterol reduction was 50% with lomitapide in its single-arm open-label registration trial. Mipomersen reduced LDL cholesterol by approximately 25% in its double-blind, placebo-controlled registration study. Both lomitapide and mipomersen therapy are associated with variable increases in hepatic fat content. The long-term safety of increased hepatic fat content in patients receiving these therapies is uncertain and requires further study. Both drugs may cause elevated transaminase in some patients, but no cases of severe liver injury have been reported. Lomitapide may also cause gastrointestinal discomfort and diarrhoea, especially if patients consume high-fat meals and patients are advised to follow a low-fat diet supplemented with essential fatty acids and fat-soluble vitamins. Mipomersen may cause injection-site and influenza-like reactions. The effect of lomitapide and mipomersen on cardiovascular outcomes has not been studied, but circumstantial evidence suggests that the LDL cholesterol lowering achieved with these two agents may reduce cardiovascular event rates.

Survival in homozygous familial hypercholesterolaemia is determined by the on-treatment level of serum cholesterol.

Aims: Homozygous familial hypercholesterolaemia (FH) is a rare inherited disorder characterized by extreme hypercholesterolaemia from birth, accelerated atherosclerosis, and premature death. Many forms of lipid-lowering therapies have been used in the past, but definitive evidence of benefit has been lacking. We therefore undertook a retrospective survey of lipid levels and clinical outcomes of FH homozygotes treated with a combination of lipid-lowering measures between 1990 and 2014 in South Africa and the UK. Methods and results: We divided 133 previously statin-naive homozygotes into quartiles according to their on-treatment levels of serum cholesterol and compared the occurrence of any death, cardiovascular death, and major adverse cardiovascular events (MACE) between the quartiles during 25 years of follow-up, using Cox and competing risks regression analysis. Patients in Quartile 4, with an on-treatment serum cholesterol >15.1 mmol/L, had a hazard ratio of 11.5 for any death compared with those in Quartile 1, with an on-treatment cholesterol of < 8.1 mmol/L. Those in Quartiles 2 and 3 combined, with on-treatment cholesterol of 8.1-15.1 mmol/L had a hazard ratio of 3.6 compared with Quartile 1. These differences were statistically significant (P < 0.001) and remained so after adjustments for confounding factors (P = 0.04). Significant differences between quartiles were also evident for cardiovascular deaths and MACE. Conclusion: These findings provide unequivocal evidence that the extent of reduction of serum cholesterol achieved by a combination of therapeutic measures, including statins, ezetimibe, lipoprotein apheresis, and evolocumab, is a major determinant of survival in homozygous FH.

Identification of a single MPV17 nonsense-associated altered splice variant in 24 South African infants with mitochondrial neurohepatopathy.

MPV17-related mitochondrial neurohepatopathy is a rare genetic disorder worldwide. We report on a novel pathogenic variant in the MPV17 gene in 24 unrelated neurohepatopathic infants of non-consanguineous Black South African heritage. Exome sequencing identified homozygosity for a c.106C>T nonsense variant in exon 3 of the human MPV17 gene in 2 unrelated index patients. mRNA analysis revealed transcripts both with and without exon 3, indicating both reduced splice efficiency and premature termination as mechanisms for disease. Carrier frequency in this population was found to be 1 in 68 (95% CI; 1/122-1/38) with an estimated newborn incidence of 1 in 18 496 (95% CI; 1/59 536-1/5776). Affected infants all presented with infantile onset neurohepatopathy with none surviving beyond infancy. This description of a relatively common pathogenic variant underlying a previously uncharacterized severe neurohepatopathy in South Africa will engender increased awareness, earlier diagnosis and possibly improve outcome if preventative or specific therapeutic options can be found.

Biological and Genetic Characterization of New and Known Necroviruses Causing an Emerging Systemic Necrosis Disease of Corn Salad (Valerianella locusta) in France.

An emerging systemic necrosis disease of corn salad was first observed in the Nantes region of France in the late 2000s. Classical virology and high-throughput sequencing approaches demonstrated that the disease is associated with four different necroviruses: tobacco necrosis virus A (TNVA), tobacco necrosis virus D (TNVD), olive mild mosaic virus (OMMV), and a novel recombinant Alphanecrovirus for which the name corn salad necrosis virus (CSNV) is proposed. Satellite tobacco necrosis virus was also frequently observed. Koch's postulates were completed for all four agents, each one alone being able to cause systemic necrosis of varying severity in corn salad. OMMV was the most frequently observed virus and causes the most severe symptoms. TNVA was the second, both in terms of prevalence and symptom severity while TNVD and CSNV were only rarely observed and caused the less severe symptoms. The emergence of this systemic disease may have been favored by the short and repeated cropping cycles used for corn salad, possibly allowing the selection of necrovirus isolates with an improved ability to systemically invade this specialty crop.

Effect of adding bezafibrate to standard lipid-lowering therapy on post-fat load lipid levels in patients with familial dysbetalipoproteinemia. A randomized placebo-controlled crossover trial.

Familial dysbetalipoproteinemia (FD) is a genetic disorder associated with impaired postprandial lipid clearance. The effect of adding bezafibrate to standard lipid-lowering therapy on postprandial and fasting lipid levels in patients with FD is unknown. In this randomized placebo-controlled double-blind crossover trial, 15 patients with FD received bezafibrate and placebo for 6 weeks in randomized order in addition to standard lipid-lowering therapy (statin, ezetimibe, and/or lifestyle). We assessed post-fat load lipids, expressed as incremental area under the curve (iAUC) and area under the curve (AUC), as well as fasting levels and safety, and found that adding bezafibrate did not reduce post-fat load non-HDL-cholesterol (non-HDL-C) iAUC (1.78 ± 4.49 mmol·h/l vs. 1.03 ± 2.13 mmol·h/l, P = 0.57), but did reduce post-fat load triglyceride (TG) iAUC (8.05 ± 3.32 mmol·h/l vs. 10.61 ± 5.92 mmol·h/l, P = 0.03) and apoB (0.64 ± 0.62 g·h/l vs. 0.93 ± 0.71 g·h/l, P = 0.01). Furthermore, bezafibrate significantly improved AUC and fasting levels of non-HDL-C, TG, total cholesterol, HDL-C, and apoB. Bezafibrate was associated with lower estimated glomerular filtration rate (78.4 ± 11.4 ml/min/1.73 m2 vs. 86.1 ± 5.85 ml/min/1.73 m2, P = 0.002). In conclusion, in patients with FD, the addition of bezafibrate to standard lipid-lowering therapy resulted in improved post-fat load and fasting plasma lipids. Combination therapy of statin/fibrate could be considered as standard lipid-lowering treatment in FD.

Marine and terrestrial foods as a source of brain-selective nutrients for early modern humans in the southwestern Cape, South Africa.

Many attempts have been made to define and reconstruct the most plausible ecological and dietary niche of the earliest members of the human species. While earlier models emphasise big-game hunting in terrestrial, largely savannah environments, more recent scenarios consider the role of marine and aquatic foods as a source of polyunsaturated fatty acids (PUFA) and other brain-selective nutrients. Along the coast of southern Africa, there appears to be an association between the emergence of anatomically modern humans and accumulation of some of the earliest shell middens during the Middle Stone Age (200-40 ka). Fragmentary fossil remains classified as those of anatomically modern humans, along with marine food residues and numerous material cultural indicators of increased social and behavioural complexity have been recovered from coastal sites. In this paper, new information on the nutrient content of marine and terrestrial foods available to early modern humans in the southwestern Cape is presented and compared with existing data on the nutritional value of some wild plant and animal foods in Africa. The results suggest that coastal foraging, particularly the collection of abundant and predictable marine molluscs, would have allowed early modern humans to exploit some of the richest and most accessible sources of protein, micronutrients and longer-chain omega-6 and omega-3 fatty acids. Reliable and accessible sources of omega-3 eicosapentaenoic and docosahexaenoic acid are considerably more restricted in terrestrial foods.

Complete Nucleotide Sequence of Artichoke latent virus Shows it to be a Member of the Genus Macluravirus in the Family Potyviridae.

Complete genomic sequences of Artichoke latent virus (ArLV) have been obtained by classical or high-throughput sequencing for an ArLV isolate from Italy (ITBr05) and for two isolates from France (FR37 and FR50). The genome is 8,278 to 8,291 nucleotides long and has a genomic organization comparable with that of Chinese yam necrotic mosaic virus (CYNMV), the only macluravirus fully sequenced to date. The cleavage sites of the viral polyprotein have been tentatively identified by comparison with CYNMV, confirming that macluraviruses are characterized by the absence of a P1 protein, a shorter and N-terminally truncated coat protein (CP). Sequence comparisons firmly place ArLV within the genus Macluravirus, and confirm previous results suggesting that Ranunculus latent virus (RALV), a previously described Macluravirus sp., is very closely related to ArLV. Serological relationships and comparisons of the CP gene and of the partial RaLV sequence available all indicate that RaLV should not be considered as a distinct species but as a strain of ArLV. The results obtained also suggest that the spectrum of currently used ArLV-specific molecular hybridization or polymerase chain reaction detection assays should be improved to cover all isolates and strains in the ArLV species.

Statin therapy reduces the mycobacterium tuberculosis burden in human macrophages and in mice by enhancing autophagy and phagosome maturation.

BACKGROUND: Statins are cholesterol-lowering drugs, targeting HMG-CoA reductase, thereby reducing the risk of coronary disorders and hypercholesterolemia. However, they also can influence immunologic responses. METHODS: Peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) were isolated from patients with familial hypercholesterolemia (FH) during statin therapy. After infection of cells with Mycobacterium tuberculosis, bacterial burden was determined. In vivo, mice were treated with statins before aerosol-based infection with M. tuberculosis and were monitored for disease progression. RESULTS: PBMCs and MDMs from patients with FH receiving statin therapy were more resistant to M. tuberculosis infection, with reduced bacterial burdens, compared with those of healthy donors. Moreover, statin treatment in experimental murine M. tuberculosis infection studies increased host protection, with reduced lung burdens and improved histopathologic findings. Mechanistically, metabolic rescue experiments demonstrated that statins reduce membrane cholesterol levels, particularly by the mevalonate-isoprenoid arm of the sterol pathway. This promoted phagosomal maturation (EEA-1/Lamp-3) and autophagy (LC3-II), as shown by confocal microscopy and Western blot in macrophages. In addition, inhibitors of phagosome and autophagosome maturation reversed the beneficial effect of statins on bacterial growth. CONCLUSION: These results suggest that statin-mediated reduction in cholesterol levels within phagosomal membranes counteract M. tuberculosis-induced inhibition of phagosomal maturation and promote host-induced autophagy, thereby augmenting host protection against tuberculosis.

Dysbetalipoproteinaemia: a mixed hyperlipidaemia of remnant lipoproteins due to mutations in apolipoprotein E.

Atherosclerosis is strongly associated with dyslipoproteinaemia, and especially with increasing concentrations of low-density lipoprotein and decreasing concentrations of high-density lipoproteins. Its association with increasing concentrations of plasma triglyceride is less clear but, within the mixed hyperlipidaemias, dysbetalipoproteinaemia (Fredrickson type III hyperlipidaemia) has been identified as a very atherogenic entity associated with both premature ischaemic heart disease and peripheral arterial disease. Dysbetalipoproteinaemia is characterized by the accumulation of remnants of chylomicrons and of very low-density lipoproteins. The onset occurs after childhood and usually requires an additional metabolic stressor. In women, onset is typically delayed until menopause. Clinical manifestations may vary from no physical signs to severe cutaneous and tendinous xanthomata, atherosclerosis of coronary and peripheral arteries, and pancreatitis when severe hypertriglyceridaemia is present. Rarely, mutations in apolipoprotein E are associated with lipoprotein glomerulopathy, a condition characterized by progressive proteinuria and renal failure with varying degrees of plasma remnant accumulation. Interestingly, predisposing genetic causes paradoxically result in lower than average cholesterol concentration for most affected persons, but severe dyslipidaemia develops in a minority of patients. The disorder stems from dysfunctional apolipoprotein E in which mutations result in impaired binding to low-density lipoprotein (LDL) receptors and/or heparin sulphate proteoglycans. Apolipoprotein E deficiency may cause a similar phenotype. Making a diagnosis of dysbetalipoproteinaemia aids in assessing cardiovascular risk correctly and allows for genetic counseling. However, the diagnostic work-up may present some challenges. Diagnosis of dysbetalipoproteinaemia should be considered in mixed hyperlipidaemias for which the apolipoprotein B concentration is relatively low in relation to the total cholesterol concentration or when there is significant disparity between the calculated LDL and directly measured LDL cholesterol concentrations. Genetic tests are informative in predicting the risk of developing the disease phenotype and are diagnostic only in the context of hyperlipidaemia. Specialised lipoprotein studies in reference laboratory centres can also assist in diagnosis. Fibrates and statins, or even combination treatment, may be required to control the dyslipidaemia.