Weight loss surgery in adolescents corrects high-density lipoprotein subspecies and their function.

BACKGROUND/OBJECTIVE: Youth with obesity have an altered high-density lipoprotein (HDL) subspecies profile characterized by depletion of large apoE-rich HDL particles and an enrichment of small HDL particles. The goal of this study was to test the hypothesis that this atherogenic HDL profile is reversible and that HDL function would improve with metabolic surgery. METHODS: Serum samples from adolescent males with severe obesity mean±s.d. age of 17.4±1.6 years were studied at baseline and 1 year following vertical sleeve gastrectomy (VSG). HDL subspecies and HDL function were evaluated pre and post VSG using paired t-tests. A lean group of adolescents was included as a reference group. RESULTS: After VSG, body mass index decreased by 32% and insulin resistance as estimated by homeostatic model assessment of insulin resistance decreased by 75% (both P<0.01). Large apoE-rich HDL subspecies increased following VSG (P<0.01) and approached that of lean adolescents despite participants with considerable residual obesity. In addition, HDL function improved compared with baseline (cholesterol efflux capacity increased by 12%, HDL lipid peroxidation potential decreased by 30% and HDL anti-oxidative capacity improved by 25%, all P<0.01). CONCLUSIONS: Metabolic surgery results in a significant improvement in the quantity of large HDL subspecies and HDL function. Our data suggest metabolic surgery may improve cardiovascular risk in adolescents and young adults.

Speciated High-Density Lipoprotein Biogenesis and Functionality.

Plasma high-density lipoprotein cholesterol (HDL-C) concentration is a negative risk factor for atherosclerotic cardiovascular disease (CVD). Despite this, most attempts to raise plasma HDL-C concentrations in a cardioprotective way have failed. Recently, hypotheses about the atheroprotective effects of HDL have shifted away from quantity to quality, mostly HDL function in reverse cholesterol transport. Plasma HDL from CVD patients is a poorer acceptor of cellular cholesterol than plasma from healthy controls, independent of plasma HDL-C concentrations. The function of HDL is likely determined by two other factors, stability and composition. The kinetic instability of HDL, which varies according to subclass, is a likely determinant of its reactivity in response to many HDL-modifying activities. HDL composition is also heterogeneous and variable; all HDL particles contain apo AI but only about two-thirds contain apo AII. This occurs despite the fact that apo AI and apo AII are hepatically secreted on separate HDL that later fuse in plasma. HDL also contains traces of other proteins, some of which have not yet been associated with HDL function. One minor HDL species are those that are secreted with intact signal peptides, which enhances their binding to HDL; these HDL have special properties that are independent of cholesterol transport. Here, we review and provide a perspective about what is currently known about speciated HDL biogenesis in the context of health and disease.

Evidence for multiple sex-determining loci in Tasmanian Atlantic salmon (Salmo salar).

Phenotypic sex in salmonids is determined primarily by a genetic male heterogametic system; yet, sex reversal can be accomplished via hormonal treatment. In Tasmanian Atlantic salmon aquaculture, to overcome problems associated with early sexual maturation in males, sex-reversed females are crossed with normal females to produce all female stock. However, phenotypic distinction of sex-reversed females (neo-males) from true males is problematic. We set out to identify genetic markers that could make this distinction. Microsatellite markers from chromosome 2 (Ssa02), to which the sex-determining locus (SEX) has been mapped in two Scottish Atlantic salmon families, did not predict sex in a pilot study of seven families. A TaqMan 64 SNP genome-wide scan suggested SEX was on Ssa06 in these families, and this was confirmed by microsatellite markers. A survey of 58 families in total representing 38 male lineages in the SALTAS breeding program found that 34 of the families had SEX on Ssa02, in 22 of the families SEX was on Ssa06, and two of the families had a third SEX locus, on Ssa03. A PCR test using primers designed from the recently published sdY gene is consistent with Tasmanian Atlantic salmon having a single sex-determining gene that may be located on at least three linkage groups.

Assessing footprints of selection in commercial Atlantic salmon populations using microsatellite data.

Relatively large rates of response to traits of economic importance have been observed in different selection experiments in salmon. Several QTL have been mapped in the salmon genome, explaining unprecedented levels of phenotypic variation. Owing to the relatively large selection intensity, individual loci may be indirectly selected, leaving molecular footprints of selection, together with increased inbreeding, as its likely relatives will share the selected loci. We used population differentiation and levels of linkage disequilibrium in chromosomes known to be harbouring QTL for body weight, infectious pancreatic necrosis resistance and infectious salmon anaemia resistance to assess the recent selection history at the genomic level in Atlantic salmon. The results clearly suggest that the marker SSA0343BSFU on chromosome 3 (body weight QTL) showed strong evidence of directional selection. It is intriguing that this marker is physically mapped to a region near the coding sequence of DVL2 , making it an ideal candidate gene to explain the rapid evolutionary response of this chromosome to selection for growth in Salmo salar. Weak evidence of diversifying selection was observed in the QTL associated with infectious pancreatic necrosis and infectious salmon anaemia resistance. Overall, this study showed that artificial selection has produced important changes in the Atlantic salmon genome, validating QTL in commercial salmon populations used for production purposes according to the recent selection history.

Mutations in MKKS cause obesity, retinal dystrophy and renal malformations associated with Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder predominantly characterized by obesity, retinal dystrophy, polydactyly, learning difficulties, hypogenitalism and renal malformations, with secondary features that include diabetes mellitus, endocrinological dysfunction and behavioural abnormalities. Despite an initial expectation of genetic homogeneity due to relative clinical uniformity, five BBS loci have been reported, with evidence for additional loci in the human genome; however, no genes for BBS have yet been identified. We performed a genome screen with BBS families from Newfoundland that were excluded from BBS1-5 and identified linkage with D20S189. Fine-mapping reduced the critical interval to 1.9 cM between D20S851 and D20S189, encompassing a chaperonin-like gene. Mutations in this gene were recently reported to be associated with McKusick-Kaufman syndrome (MKKS; ref. 8). Given both the mapping position and clinical similarities of these two syndromes, we screened MKKS and identified mutations in five Newfoundland and two European-American BBS pedigrees. Most are frameshift alleles that are likely to result in a non-functional protein. Our data suggest that a complete loss of function of the MKKS product, and thus an inability to fold a range of target proteins, is responsible for the clinical manifestations of BBS.

Adaptation genomics: next generation sequencing reveals a shared haplotype for rapid early development in geographically and genetically distant populations of rainbow trout.

Local adaptation occurs when a population evolves a phenotype that confers a selective advantage in its local environment, but which may not be advantageous in other habitats. Restricted gene flow and strong selection pressures are prerequisites for local adaptation. Fishes in the family Salmonidae are predicted to provide numerous examples of local adaptation because of the high fidelity of returning to spawn in their natal streams, which results in highly structured populations, and the wide diversity of environments that salmonids have colonized. These conditions are ideally suited for producing a set of specialist phenotypes, whose fitness is maximized for one specific habitat, rather than a generalist phenotype similarly viable in several environments. Understanding patterns and processes leading to local adaptations has long been a goal of evolutionary biology, but it is only recently that identifying the molecular basis for local adaptation has become feasible because of advances in genomic technologies. The study of shared adaptive phenotypes in populations that are both geographically distant and genetically distinct should reveal some of the fundamental molecular mechanisms associated with local adaptation. In this issue of Molecular Ecology, Miller et al. (2012) make a significant contribution to the development of adaptation genomics. This study suggests that salmonids use standing genetic variation to select beneficial alleles for local adaptations rather than de novo mutations in the same gene or alternative physiological pathways. Identifying the genetic basis for local adaptation has major implications for the management, conservation and potential restoration of salmonid populations.

Identification of olfactory receptor genes in Atlantic salmon Salmo salar.

It has been hypothesized that salmonids use olfactory cues to return to their natal rivers and streams. The key components of the molecular pathways involved in imprinting and homing, however, are still unknown. Aquatic chemical cues are received through the nares and into the nasal cavity that contains a single olfactory organ, the olfactory rosette. The olfactory rosette contains sensory neurons, each of which is thought to express only one olfactory receptor. If odorants are involved in salmonid homing migration then olfactory receptors should play a critical role in the dissipation of information from the environment to the fish. Therefore, to understand the molecular basis for imprinting and homing in Atlantic salmon Salmo salar it is important to identify and characterize the repertoire of olfactory receptors in this species. The first public assembly of the S. salar genome was searched for genes encoding three of the superfamilies of fish olfactory receptors: V2R-like (olfc), V1R-like (ora) and main olfactory receptor (mor). A further six ora genes were added to ora1 and ora2, which had been described previously. In addition, 48 putative mors were identified, 24 of which appear to be functional based on their gene structures and predicted amino-acid sequences. Phylogenetic analyses were then used to compare these S. salar olfactory receptor genes with those of zebrafish Danio rerio, two pufferfish species Takifugu rubripes and Tetraodon nigroviridis, medaka Oryzias latipes and three-spined stickleback Gasterosteus aculeatus.

Identification of the sex chromosomes of brown trout (Salmo trutta) and their comparison with the corresponding chromosomes in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss).

Males are the heterogametic sex in salmonid fishes. In brown trout (Salmo trutta) the sex-determining locus, SEX, has been mapped to the end of linkage group BT-28, which corresponds to linkage group AS-8 and chromosome SSA15 in Atlantic salmon (Salmo salar). We set out to identify the sex chromosomes in brown trout. We isolated Atlantic salmon BAC clones containing microsatellite markers that are on BT-28 and also on AS-8, and used these BACs as probes for fluorescent in situ hybridization (FISH) analysis. SEX is located on the short arm of a small subtelocentric/acrocentric chromosome in brown trout, which is consistent with linkage analysis. The acrocentric chromosome SSA15 in Atlantic salmon appears to have arisen by a centric fusion of 2 small acrocentric chromosomes in the common ancestor of Salmo sp. We speculate that the fusion process that produced Atlantic salmon chromosome SSA15 disrupted the ancestral sex-determining locus in the Atlantic salmon lineage, providing the impetus either for the relocation of SEX or selection pressure for a novel sex-determining gene to arise in this species. Thus, the sex-determining genes may differ in Atlantic salmon and brown trout.

Expression of olfactory receptors in different life stages and life histories of wild Atlantic salmon (Salmo salar).

It has been hypothesized that salmonids use olfactory cues to return to their natal rivers and streams. However, the key components of the molecular pathway involved in imprinting and homing are still unknown. If odorants are involved in salmon homing migration, then olfactory receptors should play a critical role in the dissipation of information from the environment to the fish. Therefore, we examined the expression profiles of a suite of genes encoding olfactory receptors and other olfactory-related genes in the olfactory rosettes of different life stages in two anadromous and one non-anadromous wild Atlantic salmon populations from Newfoundland, Canada. We identified seven differentially expressed OlfC genes in juvenile anadromous salmon compared to returning adults in both populations of anadromous Atlantic salmon. The salmon from the Campbellton River had an additional 10 genes that were differentially expressed in juveniles compared to returning adults. There was no statistically significant difference in gene expression of any of the genes in the non-anadromous population (P < 0.01). The function of the OlfC gene products is not clear, but they are predicted to be amino acid receptors. Other studies have suggested that salmon use amino acids for imprinting and homing. This study, the first to examine the expression of olfactory-related genes in wild North American Atlantic salmon, has identified seven OlfC genes that may be involved in the imprinting and homeward migration of anadromous Atlantic salmon.

BAC-based upgrading and physical integration of a genetic SNP map in Atlantic salmon.

A better understanding of the genotype-phenotype correlation of Atlantic salmon is of key importance for a whole range of production, life history and conservation biology issues attached to this species. High-density linkage maps integrated with physical maps and covering the complete genome are needed to identify economically important genes and to study the genome architecture. Linkage maps of moderate density and a physical bacterial artificial chromosome (BAC) fingerprint map for the Atlantic salmon have already been generated. Here, we describe a strategy to combine the linkage mapping with the physical integration of newly identified single nucleotide polymorphisms (SNPs). We resequenced 284 BAC-ends by PCR in 14 individuals and detected 180 putative SNPs. After successful validation of 152 sequence variations, genotyping and genetic mapping were performed in eight salmon families comprising 376 individuals. Among these, 110 SNPs were positioned on a previously constructed linkage map containing SNPs derived from expressed sequence tag (EST) sequences. Tracing the SNP markers back to the BACs enabled the integration of the genetic and physical maps by assigning 73 BAC contigs to Atlantic salmon linkage groups.

Grayling (Thymallinae) phylogeny within salmonids: complete mitochondrial DNA sequences of Thymallus arcticus and Thymallus thymallus.

The phylogenetic relationships among the three subfamilies (Salmoninae, Coregoninae and Thymallinae) in the Salmonidae have not been addressed extensively at the molecular level. In this study, the whole mitochondrial genomes of two Thymallinae species, Thymallus arcticus and Thymallus thymallus were sequenced, and the published mitochondrial genome sequences of other salmonids were used for Bayesian and maximum-likelihood phylogenetic analyses. These results support an ancestral Coregoninae, branching within the Salmonidae, with Thymallinae as the sister group to Salmoninae.

Triallelic inheritance in Bardet-Biedl syndrome, a Mendelian recessive disorder.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder characterized by multiple clinical features that include pigmentary retinal dystrophy, polydactyly, obesity, developmental delay, and renal defects. BBS is considered an autosomal recessive disorder, and recent positional cloning efforts have identified two BBS genes (BBS2 and BBS6). We screened our cohort of 163 BBS families for mutations in both BBS2 and BBS6 and report the presence of three mutant alleles in affected individuals in four pedigrees. In addition, we detected unaffected individuals in two pedigrees who carry two BBS2 mutations but not a BBS6 mutation. We therefore propose that BBS may not be a single-gene recessive disease but a complex trait requiring three mutant alleles to manifest the phenotype. This triallelic model of disease transmission may be important in the study of both Mendelian and multifactorial disorders.

Rainbow trout interleukin-2: cloning, expression and bioactivity analysis.

In this study the rainbow trout (Oncorhynchus mykiss) interleukin-2 (IL-2) cDNA has been cloned, and its expression and bioactivity analysed in head kidney leucocytes. The IL-2 precursor encoded an open reading frame of 429 bp, that translates into a predicted protein of 142 aa, with a 20 aa signal peptide. The trout IL-2 had moderate protein homology (30.9% identity/48.3% similarity) with Fugu IL-2, the only IL-2 homologue identified in fish to date, with lower homology to avian (17.8% identity/23.2% similarity) and mammalian (34.2 identity/46.5% similarity) IL-2s. IL-2 expression was induced by the T cell mitogen PHA and by the mixed leucocyte reaction, where leucocytes from pairs of fish were cultured together for four days. Expression was also induced in vivo during bacterial (Yersinia ruckeri) infection. The Escherichia coli produced recombinant IL-2 was shown to increase the expression of two transcription factors, STAT5 and Blimp-1, known to be involved in IL-2 signalling in mammals, as well as IFN-gamma, gIP and IL-2 itself. The potential signalling pathways involved and possible use as an adjuvant for fish vaccines are discussed.

Rainbow smelt (Osmerus mordax) genomic library and EST resources.

Genomic resources in rainbow smelt (Osmerus mordax) enable us to examine the genome duplication process in salmonids and test hypotheses relating to the fate of duplicated genes. They further enable us to pursue physiological and ecological studies in smelt. A bacterial artificial chromosome library containing 52,410 clones with an average insert size of 146 kb was constructed. This library represents an 11-fold average coverage of the rainbow smelt (O. mordax) genome. In addition, several complementary deoxyribonucleic acid libraries were constructed, and 36,758 sequences were obtained and combined into 12,159 transcripts. Over half of these transcripts have been identified, several of which have been associated with cold adaptation. These basic resources show high levels of similarity (86%) to salmonid genes and provide initial support for genome duplication in the salmonid ancestor. They also facilitate identification of genes important to fish and direct us toward new technologies for other studies in fish biology.

Identification of the sex-determining locus of Atlantic salmon (Salmo salar) on chromosome 2.

We have integrated data from linkage mapping, physical mapping and karyotyping to gain a better understanding of the sex-determining locus, SEX, in Atlantic salmon (Salmo salar). SEX has been mapped to Atlantic salmon linkage group 1 (ASL1) and is associated with several microsatellite markers. We have used probes designed from the flanking regions of these sex-linked microsatellite markers to screen a bacterial artificial chromosome (BAC) library, representing an 11.7x coverage of the Atlantic salmon genome, which has been HindIII fingerprinted and assembled into contigs. BACs containing sex-linked microsatellites and their related contigs have been identified and representative BACs have been placed on the Atlantic salmon chromosomes by fluorescent in situ hybridization (FISH). This identified chromosome 2, a large metacentric, as the sex chromosome. By positioning several BACs on this chromosome by FISH, it was possible to orient ASL1 with respect to chromosome 2. The region containing SEX appears to lie on the long arm between marker Ssa202DU and a region of heterochromatin identified by DAPI staining. BAC end-sequencing of clones within sex-linked contigs revealed five hitherto unmapped genes along the sex chromosome. We are using an in silico approach coupled with physical probing of the BAC library to extend the BAC contigs to provide a physical map of ASL1, with a view to sequencing chromosome 2 and, in the process, identifying the sex-determining gene.

Evidence of recent signatures of selection during domestication in an Atlantic salmon population.

Selective breeding practices in Atlantic salmon aquaculture have been carried out intensively since the 1970s. Along with the phenotypic improvement of fish, we expect to observe genomic regions showing evidence of selection for traits related to growth and age at sexual maturation, as well as traits involved in the domestication process. This is mainly linked to the increase in the frequency of favourable alleles at loci that affect the traits of interest in the breeding population. In this study we searched for signatures of selection in the Cermaq Atlantic salmon broodstock, a Mowi strain, which was derived from wild Norwegian populations, and is now farmed in British Columbia, Canada. A 6.5K SNP array was used to genotype 202 fish from the Cermaq population, and the genotypes were compared with four wild populations from Norway. We used three methods based on FST values to detect signatures of selection. Forty four markers showing divergence in allele frequency were identified as outliers by the three detection methods, suggesting the presence of signatures of selection in the Cermaq population relative to their wild counterparts. Markers identified as outliers are associated with molecular functions that could be related to the selection for economically important traits (e.g., growth) as well as the domestication process (e.g., response to pathogens and environmental stressors). Of particular interest were three outlier markers that had been previously associated with grilsing (i.e., early sexual maturation) an undesirable trait, which has been heavily selected against in Atlantic salmon aquaculture. This study provides clear evidence of the presence of signatures of selection and domestication in a farmed Atlantic salmon population.

Examination of the peptide sequence requirements for lipid-binding. Alternative pathways for promoting the interaction of amphipathic alpha-helical peptides with phosphatidylcholine.

To examine the relationship between peptide sequence and the interaction of amphipathic alpha-helical peptides with phosphatidylcholines, various methods of mixing the peptide and lipid were explored. A series of amphipathic alpha-helical peptides containing from 10 to 18 residues were synthesized by solid-phase techniques. An 18-residue peptide and two relatively hydrophobic 10-residue peptides did not disrupt dimyristoylphosphatidylcholine liposomes when added to the lipid in buffer. However, when the peptides were premixed with lipid in a suitable organic solvent and then reconstituted with aqueous buffer, clear micelles were formed, indicating association of the amphipathic alpha-helical peptide with lipid. In general, the best solvent for this purpose was trifluoroethanol. The circular dichroic and fluorescence spectra of peptides which readily formed clear mixtures when mixed in buffer with dimyristoylphosphatidylcholine liposomes were similar when prepared either by the alternative pathway technique using trifluoroethanol or by a cholate removal technique. For the peptides which did not clear liposomes in buffer, first mixing with dimyristoylphosphatidylcholine in trifluoroethanol resulted in an increase in the alpha-helicity of the peptides as judged by circular dichroic spectra and a blue-shift in the fluorescence emission maxima of the single tryptophan residue in each peptide. These data are consistent with formation of an amphipathic alpha-helix in lipid by peptides which based on mixing experiments with dimyristoylphosphatidylcholine liposomes in buffer at the phase transition temperature of the lipid would be considered ineffective in lipid binding. Thus, simple mixing of peptides with liposomes may give misleading results concerning the intrinsic affinity of a particular peptide sequence for lipid. In addition, the data demonstrate that relatively hydrophobic amphipathic alpha-helical peptides which do not form small micelles with dimyristoylphosphatidylcholine spontaneously in aqueous solution may interact with lipid as typical amphipathic alpha-helices when mixed by an alternative pathway.

Tc1 transposon-like sequences are widely distributed in salmonids.

Transposon-like elements flanked by inverted repeats, although common in invertebrates, have only recently been found in vertebrates. We report the presence of Tc1 transposon-like sequences in salmon, trout and charr species and find that these elements belong to several families that do not follow phylogenetic lines. As many as 15,000 copies reside in the Atlantic salmon haploid genome. The complete DNA sequence of one of these transposon-like elements (SALT1) is 1535 base-pairs long, including 35 nucleotide-long terminal inverted repeats. It contains a degenerate open reading frame (ORF) of 1273 nucleotides whose inferred amino acid sequence shares sequence similarity with the "D,D35E" family of transposases, particularly those from Caenorhabditis sp. and Drosophila sp. Southern blot analysis indicated that Tc1 transposon-like sequences are present in other lower vertebrates, including several fish species and amphibians, but the copy number can vary significantly in different lineages.

A repetitive element in the genome of Atlantic salmon, Salmo salar.

When Atlantic salmon (Salmo salar) genomic DNA is digested with the restriction endonuclease BglI and the fragments separated by agarose-gel electrophoresis, bands corresponding to approximately 430 and 923 bp are visualized after EtdBr staining. The 923-bp band was excised from a preparative gel and used to screen a salmon genomic library for recombinant phage (re-phage) containing the repeat. The BglI repeat element is tandemly arrayed, and an array from one re-phage has been sequenced. The BglI repeats comprise 2.3% of the S. salar genome and have been found in the vicinity of rDNA genes (encoding ribosomal RNA). Southern blot hybridization detects a homologue of the Atlantic salmon BglI repeat in the brown trout (Salmo trutta) genome, but not in other salmonids. However, a DNA fragment with sequence homology to part of the BglI repeat has recently been isolated from Arctic charr (Salvelinus alpinus; S.E. Hartley and W.S.D., unpublished data). In addition, the BglI repeat detects RFLPs in Atlantic salmon.

The complete mitochondrial DNA sequence of the Atlantic salmon, Salmo salar.

The complete sequence of the Atlantic salmon (Salmo salar) mitochondrial genome has been determined. The entire sequence is 16665 base pairs (bp) in length, with a gene content (13 protein-coding, two ribosomal RNA [rRNA] and 22 transfer RNA [tRNA] genes) and order conforming to that observed in most other vertebrates. Base composition and codon usage have been detailed. Nucleotide and derived amino acid sequences of the 13 protein-coding genes from Atlantic salmon have been compared with their counterparts in rainbow trout. A putative structure for the origin of L-strand replication (O(L)) is proposed, and sequence features of the control region (D-loop) are described.