Early onset collagen VI myopathies: Genetic and clinical correlations.

OBJECTIVE: Mutations in the genes encoding the extracellular matrix protein collagen VI (ColVI) cause a spectrum of disorders with variable inheritance including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate phenotypes. We extensively characterized, at the clinical, cellular, and molecular levels, 49 patients with onset in the first 2 years of life to investigate genotype-phenotype correlations. METHODS: Patients were classified into 3 groups: early-severe (18%), moderate-progressive (53%), and mild (29%). ColVI secretion was analyzed in patient-derived skin fibroblasts. Chain-specific transcript levels were quantified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and mutation identification was performed by sequencing of complementary DNA. RESULTS: ColVI secretion was altered in all fibroblast cultures studied. We identified 56 mutations, mostly novel and private. Dominant de novo mutations were detected in 61% of the cases. Importantly, mutations causing premature termination codons (PTCs) or in-frame insertions strikingly destabilized the corresponding transcripts. Homozygous PTC-causing mutations in the triple helix domains led to the most severe phenotypes (ambulation never achieved), whereas dominant de novo in-frame exon skipping and glycine missense mutations were identified in patients of the moderate-progressive group (loss of ambulation). INTERPRETATION: This work emphasizes that the diagnosis of early onset ColVI myopathies is arduous and time-consuming, and demonstrates that quantitative RT-PCR is a helpful tool for the identification of some mutation-bearing genes. Moreover, the clinical classification proposed allowed genotype-phenotype relationships to be explored, and may be useful in the design of future clinical trials.

Molecular cophylogenetic relationships between European bats and their ectoparasitic mites (Acari, Spinturnicidae).

Cospeciation between host-parasite species is generally thought to result in mirror-image congruent phylogenies. Incongruence can be explained by mechanisms such as host switching, duplication, failure to speciate and sorting events. To investigate the level of association in the host-parasite relationship between Spinturnicid mites and their bat hosts, we constructed the phylogenetic tree of the genus Spinturnix (Acari, Mesostigmata) and compared it to the host phylogeny. We sequenced 938bp of the mitochondrial 16S rDNA and Cytochrome Oxydase subunit I (COI) genes among eleven morphospecies of Spinturnix collected on 20 European Vespertilionid and Rhinolophid bat species. Phylogenetic reconstruction of hosts and parasites showed statistical evidence for cospeciation and suggested that their evolutionary history involved also failure to speciate events and host switches. The latter seem to be mainly promoted by similar roosting habits of the host. As currently understood, host associations of Spinturnicid mites likely results from a complex interaction between the phylogenetic history of the host and the behaviour and the ecology of both parasite and host.

Molecular phylogeography of the nose-horned viper (Vipera ammodytes, Linnaeus (1758)): evidence for high genetic diversity and multiple refugia in the Balkan peninsula

The nose-horned viper (Vipera ammodytes) occurs in a large part of the south-eastern Europe and Asia Minor. Phylogenetic relationships were reconstructed for a total of 59 specimens using sequences from three mitochondrial regions (16S and cytochrome b genes, and control region, totalling 2308 bp). A considerable number of clades were observed within this species, showing a large genetic diversity within the Balkan peninsula. Splitting of the basal clades was evaluated to about 4 million years ago. Genetic results are in contradiction with presently accepted taxonomy based on morphological characters: V. a. gregorwallneri and V. a. ruffoi do not display any genetic difference compared with the nominotypic subspecies (V. a. ammodytes), involving that these subspecies can be regarded as synonyms. High genetic divergence in the central part of the Balkan peninsula is not concordant with low morphological differentiation. Finally, the extensive genetic diversity within the Balkan peninsula and the colonisation routes are discussed

Systematic and biogeographic relationships of the Japanese white-toothed shrew (Crocidura dsinezumi)

To assess the zoogeographic and phylogenetic relationships of C. dsinezumi, its systematic position has been investigated through electrophoretic comparisons. These comparisons comprise typical Indomalayan and Palaearctic species, including C. russula, the supposed sister taxon of the Japanese white-toothed shrew. The range of measured genetic distances that separate dsinezumi from the other shrews (Nei's D = 0.232-0.406) is typical for interspecific comparisons among Crocidura taxa. Our results reinforce morphologic considerations that suggest C. dsinezumi be treated as a valid species. Phylogenetic relationships deduced from a cladistic treatment of allozyme data refute the possibility that the western Palaearctic C. russula is the sister taxon of C. dsinezumi. Zoogeographic interpretation of the genetic results indicates that the Southeast Asian representatives constitute a clade distinct from one that includes the Palaearctic species. However, C. dsinezumi is difficult to assign to either clade due to many intermediate or plesiomorphic electrophoretic characters. Unlike the major karyologic and biochemical separation that divides Afrotropical and Palaearctic species, the subdivision between Palaearctic and Indomalayan Crocidura is not correlated with any trend in karyotypic evolution. It also is shown that the Eurasiatic species of Crocidura possessing 2n = 40 chromosomes (including C. dsinezumi) do not represent a particular clade, but probably share a plesiomorphic character. Further analysis of other Indian or Asiatic species is needed to test whether the Palaearctic versus Indomalayan separation is due to real zoogeographic barriers or whether it is the result of limited samples.

Prediction of cross-recognition of peptide-HLA A2 by Melan-A-specific cytotoxic T lymphocytes using three-dimensional quantitative structure-activity relationships.

The cross-recognition of peptides by cytotoxic T lymphocytes is a key element in immunology and in particular in peptide based immunotherapy. Here we develop three-dimensional (3D) quantitative structure-activity relationships (QSARs) to predict cross-recognition by Melan-A-specific cytotoxic T lymphocytes of peptides bound to HLA A*0201 (hereafter referred to as HLA A2). First, we predict the structure of a set of self- and pathogen-derived peptides bound to HLA A2 using a previously developed ab initio structure prediction approach [Fagerberg et al., J. Mol. Biol., 521-46 (2006)]. Second, shape and electrostatic energy calculations are performed on a 3D grid to produce similarity matrices which are combined with a genetic neural network method [So et al., J. Med. Chem., 4347-59 (1997)] to generate 3D-QSAR models. The models are extensively validated using several different approaches. During the model generation, the leave-one-out cross-validated correlation coefficient (q (2)) is used as the fitness criterion and all obtained models are evaluated based on their q (2) values. Moreover, the best model obtained for a partitioned data set is evaluated by its correlation coefficient (r = 0.92 for the external test set). The physical relevance of all models is tested using a functional dependence analysis and the robustness of the models obtained for the entire data set is confirmed using y-randomization. Finally, the validated models are tested for their utility in the setting of rational peptide design: their ability to discriminate between peptides that only contain side chain substitutions in a single secondary anchor position is evaluated. In addition, the predicted cross-recognition of the mono-substituted peptides is confirmed experimentally in chromium-release assays. These results underline the utility of 3D-QSARs in peptide mimetic design and suggest that the properties of the unbound epitope are sufficient to capture most of the information to determine the cross-recognition.

Genetic differentiation and zoogeography of Asian Suncus murinus (Mammalia: Soricidae)

Protein electrophoresis was used to assess the phylogenetic relationships of populations of the phenotypically variable Asian house shrew Suncus murinus. These populations represent a sample of both commensal and wild forms. They were compared to another taxon, S. montanus, which was formerly considered conspecific with S. murinus. Suncus dayi was used as an outgroup in all phylogenetic reconstructions. Within the S. murinus lineage, the allozyme data show very low levels of genetic differentiation among both wild and commensal Southeast Asian and Japanese samples when compared to the Indian populations. This pattern is consistent with the classical hypothesis of a recent introduction by man in Eastern Asia. The higher genetic diversity found within S. murinus from India, as well as previous mitochondrial and karyological results suggest that this area is the probable centre of origin for the species. Although the lack of gene flow between S. murinus and S. montanus is clearly established in an area of sympatry in Southern India, one Asian house shrew sampled in Nepal was more closely related to S. montanus. This could either reflect the retention of an ancestral polymorphism, or result from a hybridization episode between S. murinus and S. montanus. Similar conclusions were also suggested in mitochondrial DNA studies dealing with animals sampled in the Northern parts of the Indian subcontinent. Clearly, further data on Suncus from this area are needed in order to assess these hypotheses. (C) 1995 The Linnean Society of London

Relationships among endogenous ouabain, alpha-adducin polymorphisms and renal sodium handling in primary hypertension.

OBJECTIVE: The basolateral Na pump drives renotubular reabsorption. In cultured renal cells, mutant adducins, as well as sub-nanomolar ouabain concentrations, stimulate the Na-K pump. METHODS: To determine whether these factors interact and affect Na handling and blood pressure (BP) in vivo, we studied 155 untreated hypertensive patients subdivided on the basis of their plasma endogenous ouabain or alpha-adducin genotype (ADD1 Gly460Trp-rs4961). RESULTS: Under basal conditions, proximal tubular reabsorption and plasma Na were higher in patients with mutated Trp ADD1 or increased endogenous ouabain (P = 0.002 and 0.05, respectively). BPs were higher in the high plasma endogenous ouabain group (P = 0.001). Following volume loading, the increment in BP (7.73 vs. 4.81 mmHg) and the slopes of the relationship between BP and Na excretion were greater [0.017 +/- 0.002 vs. 0.009 +/- 0.003 mmHg/(muEq min)] in ADD1 Trp vs. ADD1 Gly carriers (P < 0.05). BP changes were similar, whereas the slopes of the relationship between BP and Na excretion were lower [0.016 +/- 0.003 vs. 0.008 +/- 0.002 mmHg/(muEq min)] in patients with low vs. high endogenous ouabain (P < 0.05). In patients with high endogenous ouabain, volume loading increased the BP in the ADD1 Trp group but not in the Gly group (P < 0.05). Thus, patients with ADD1 Trp alleles are sensitive to salt and tubular Na reabsorption remains elevated after volume expansion. CONCLUSION: With saline loading, BP changes are similar in high and low endogenous ouabain patients, whereas tubular Na reabsorption increases in the high endogenous ouabain group. Saline loading unmasks differences in renal Na handling in patients with mutant adducin or high endogenous ouabain and exposes an interaction of endogenous ouabain and Trp alleles on BP.

Genetic association analyses reveal a role for vitamin D insufficiency in hepatitis C virus-associated hepatocellular carcinoma development

Background: V itamin D insufficiency has been associated with the occurrence of various types of cancer, but causal relationships remain elusive. Methods: Associations between t he r isk o f HCV-related HCC development and CYP2R1 , GC, and DHCR7 genotypes, which are genetic determinants of reduced 25-OH-vitamin D3 (25[OH]D3) serum levels, were determined. Results: A t otal of 5604 HCV-infected patients, 1279 with a nd 4325 without progression to HCC, w ere identified. The well-known association between 25(OH)D3 s erum levels and variations in CYP2R1 ( rs1993116, rs10741657), GC ( rs2282679), a nd DHCR7 ( rs7944926, rs12785878) g enotypes was also apparent in patients w ith chronic hepatitis C. The same genotypes of t hese single nucleotide polymorphisms (SNPs), w hich are associated with reduced 25(OH)D3 s erum levels, were significantly associated with HCV-associated HCC (P=0.07 [OR=1.13] for CYP2R1 , P=0.007 [OR=1.56] for GC, P=0.003 [OR=1.42] for DHCR7; ORs for risk genotypes). In contrast, no association between t hese genetic variations and the o utcome of antiviral therapy with pegylated interferon-α and ribavirin ( P>0.2 for e ach SNP) or liver fibrosis progression rate (P>0.2 for each SNP) was observed, s uggesting a specific influence o f the genetic d eterminants of 25(OH)D3 s erum levels o n hepatocarcinogenesis. Conclusions: Our data suggest a relatively weak but functionally relevant role for vitamin D in the prevention of HCV-related HCC development. Controlled clinical trials to assess the benefit of vitamin D supplementation in HCVinfected patients with advanced liver fibrosis or cirrhosis are warranted.

Past climate-driven range shifts and population genetic diversity in arctic plants

AimHigh intra-specific genetic diversity is necessary for species adaptation to novel environments under climate change, but species tracking suitable conditions are losing alleles through successive founder events during range shift. Here, we investigated the relationship between range shift since the Last Glacial Maximum (LGM) and extant population genetic diversity across multiple plant species to understand variability in species responses. LocationThe circumpolar Arctic and northern temperate alpine ranges. MethodsWe estimated the climatic niches of 30 cold-adapted plant species using range maps coupled with species distribution models and hindcasted species suitable areas to reconstructions of the mid-Holocene and LGM climates. We computed the species-specific migration distances from the species glacial refugia to their current distribution and correlated distances to extant genetic diversity in 1295 populations. Differential responses among species were related to life-history traits. ResultsWe found a negative association between inferred migration distances from refugia and genetic diversities in 25 species, but only 11 had statistically significant negative slopes. The relationships between inferred distance and population genetic diversity were steeper for insect-pollinated species than wind-pollinated species, but the difference among pollination system was marginally independent from phylogenetic autocorrelation. Main conclusionThe relationships between inferred migration distances and genetic diversities in 11 species, independent from current isolation, indicate that past range shifts were associated with a genetic bottleneck effect with an average of 21% loss of genetic diversity per 1000km(-1). In contrast, the absence of relationship in many species also indicates that the response is species specific and may be modulated by plant pollination strategies or result from more complex historical contingencies than those modelled here.

Restriction site-associated DNA sequencing, genotyping error estimation and de novo assembly optimization for population genetic inference.

Restriction site-associated DNA sequencing (RADseq) provides researchers with the ability to record genetic polymorphism across thousands of loci for nonmodel organisms, potentially revolutionizing the field of molecular ecology. However, as with other genotyping methods, RADseq is prone to a number of sources of error that may have consequential effects for population genetic inferences, and these have received only limited attention in terms of the estimation and reporting of genotyping error rates. Here we use individual sample replicates, under the expectation of identical genotypes, to quantify genotyping error in the absence of a reference genome. We then use sample replicates to (i) optimize de novo assembly parameters within the program Stacks, by minimizing error and maximizing the retrieval of informative loci; and (ii) quantify error rates for loci, alleles and single-nucleotide polymorphisms. As an empirical example, we use a double-digest RAD data set of a nonmodel plant species, Berberis alpina, collected from high-altitude mountains in Mexico.

Impact of genetic SLC28 transporter and ITPA variants on ribavirin 21 serum level, hemoglobin drop and therapeutic response in patients with HCV infection

Background: T reatment o f chronic hepatitis C i s evolving, a nd direct acting antivirals ( DAAs) are now a dded to p egylated interferon-α ( Peg- INF-α) and ribavirin (RBV) for the treatment o f hepatitis C v irus ( HCV) genotype 1 infection. DAAs c ause d ifferent side effects and can even worsen RBV induced hemolytic anemia. T herefore, identifying host genetic d eterminants of R BV bioavailability and therapeutic e fficacy will remain crucial for individualized treatment. Recent d ata showed associations between R BV induced h emolytic anemia and genetic polymorphisms o f concentrative nucleoside transporters s uch as C NT3 (SLC28A3) and i nosine t riphosphatase (ITPA). T o analyze t he association of genetic variants of SLC28 transporters and ITPA with RBV induced hemolytic anemia and treatment o utcome. Methods: I n our study, 173 patients f rom t he S wiss Hepatitis C C ohort Study and 2 2 patients from Swiss Association for the Study of the Liver study 24 (61% HCV g enotype 1, 3 9% genotypes 2 o r 3) were analyzed for SLC28A2 single nucleotide p olymorphism (SNP) rs11854484, SLC28A3 rs56350726 and SLC28A3 rs10868138 as well as ITPA SNPs rs1127354 and rs7270101. RBV serum levels during treatment were measured in 49 patients. Results: SLC28A2 r s11854484 genotype TT was associated with significantly higher dosage- and body weight-adjusted RBV levels as compared to genotypes TC and CC (p=0.04 and p=0.02 at weeks 4 and 8, respectively). ITPA SNPs rs1127354 and rs7270101 were associated with h emolytic a nemia both in genotype as w ell as i n allelic a nalyses. SLC28A3 rs56350726 genotype TT (vs. AT/AA, RR=2.1; 95% CI 1.1-4.1) as well as the T allele (vs. A; RR=1.8, 95% CI 1.1-3.2) were associated with increased SVR rates. The combined analysis of overall ITPA activity and SLC28 v ariants together revealed n o significant a dditive effects on either treatment-related anemia or SVR. Conclusions: T he newly identified association between RBV serum levels a nd SLC28A2 rs11854484 genotype as well as the replicated association of ITPA and SLC28A3 g enetic p olymorphisms w ith RBV induced hemolytic anemia and treatment r esponse underpin the need for further studies on host genetic d eterminants of R BV bioavailability and therapeutic e fficacy f or individualized treatment of chronic hepatitis C.

Mediterranean populations of the lesser white-toothed shrew (Crocidura suaveolens group): an unexpected puzzle of Pleistocene survivors and prehistoric introductions.

An earlier study revealed the strong phylogeographical structure of the lesser white-toothed shrew (Crocidura suaveolens group) within the northern Palaearctic. Here, we aim to reconstruct the colonization history of Mediterranean islands and to clarify the biogeography and phylogeographical relationships of the poorly documented Middle East region with the northern Palaearctic. We performed analyses on 998-bp-long haplotypes of the mitochondrial cytochrome b gene of 143 samples collected around the Mediterranean basin, including islands and the Middle East. The analyses suggest that the Cypriot shrew belongs to the rare group of relict insular Pleistocene mammal taxa that have survived to the present day. In contrast, the Cretan, Corsican and Menorcan populations were independently introduced from the Middle East during the Holocene. The phylogeographical structure of this temperate Palaearctic species within the Middle East appears to be complex and rich in diversity, probably reflecting fragmentation of the area by numerous mountain chains. Four deeply divergent clades of the C. suaveolens group occur in the area, meaning that a hypothetical contact zone remains to be located in central western Iran.

Risk prediction of prevalent diabetes in a Swiss population using a weighted genetic score--the CoLaus Study.

AIMS/HYPOTHESIS: Several susceptibility genes for type 2 diabetes have been discovered recently. Individually, these genes increase the disease risk only minimally. The goals of the present study were to determine, at the population level, the risk of diabetes in individuals who carry risk alleles within several susceptibility genes for the disease and the added value of this genetic information over the clinical predictors. METHODS: We constructed an additive genetic score using the most replicated single-nucleotide polymorphisms (SNPs) within 15 type 2 diabetes-susceptibility genes, weighting each SNP with its reported effect. We tested this score in the extensively phenotyped population-based cross-sectional CoLaus Study in Lausanne, Switzerland (n = 5,360), involving 356 diabetic individuals. RESULTS: The clinical predictors of prevalent diabetes were age, BMI, family history of diabetes, WHR, and triacylglycerol/HDL-cholesterol ratio. After adjustment for these variables, the risk of diabetes was 2.7 (95% CI 1.8-4.0, p = 0.000006) for individuals with a genetic score within the top quintile, compared with the bottom quintile. Adding the genetic score to the clinical covariates improved the area under the receiver operating characteristic curve slightly (from 0.86 to 0.87), yet significantly (p = 0.002). BMI was similar in these two extreme quintiles. CONCLUSIONS/INTERPRETATION: In this population, a simple weighted 15 SNP-based genetic score provides additional information over clinical predictors of prevalent diabetes. At this stage, however, the clinical benefit of this genetic information is limited.

Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study.

BACKGROUND & AIMS: Hepatitis C virus (HCV) induces chronic infection in 50% to 80% of infected persons; approximately 50% of these do not respond to therapy. We performed a genome-wide association study to screen for host genetic determinants of HCV persistence and response to therapy. METHODS: The analysis included 1362 individuals: 1015 with chronic hepatitis C and 347 who spontaneously cleared the virus (448 were coinfected with human immunodeficiency virus [HIV]). Responses to pegylated interferon alfa and ribavirin were assessed in 465 individuals. Associations between more than 500,000 single nucleotide polymorphisms (SNPs) and outcomes were assessed by multivariate logistic regression. RESULTS: Chronic hepatitis C was associated with SNPs in the IL28B locus, which encodes the antiviral cytokine interferon lambda. The rs8099917 minor allele was associated with progression to chronic HCV infection (odds ratio [OR], 2.31; 95% confidence interval [CI], 1.74-3.06; P = 6.07 x 10(-9)). The association was observed in HCV mono-infected (OR, 2.49; 95% CI, 1.64-3.79; P = 1.96 x 10(-5)) and HCV/HIV coinfected individuals (OR, 2.16; 95% CI, 1.47-3.18; P = 8.24 x 10(-5)). rs8099917 was also associated with failure to respond to therapy (OR, 5.19; 95% CI, 2.90-9.30; P = 3.11 x 10(-8)), with the strongest effects in patients with HCV genotype 1 or 4. This risk allele was identified in 24% of individuals with spontaneous HCV clearance, 32% of chronically infected patients who responded to therapy, and 58% who did not respond (P = 3.2 x 10(-10)). Resequencing of IL28B identified distinct haplotypes that were associated with the clinical phenotype. CONCLUSIONS: The association of the IL28B locus with natural and treatment-associated control of HCV indicates the importance of innate immunity and interferon lambda in the pathogenesis of HCV infection.

A simple genetic basis for complex social behaviour mediates widespread gene expression differences.

A remarkable social polymorphism is controlled by a single Mendelian factor in the fire ant Solenopsis invicta. A genomic element marked by the gene Gp-9 determines whether workers tolerate one or many fertile queens in their colony. Gp-9 was recently shown to be part of a supergene with two nonrecombining variants, SB and Sb. SB/SB and SB/Sb queens differ in how they initiate new colonies, and in many physiological traits, for example odour and maturation rate. To understand how a single genetic element can affect all these traits, we used a microarray to compare gene expression patterns between SB/SB and SB/Sb queens of three different age classes: 1-day-old unmated queens, 11-day-old unmated queens and mated, fully reproductive queens collected from mature field colonies. The number of genes that were differentially expressed between SB/SB and SB/Sb queens of the same age class was smallest in 1-day-old queens, maximal in 11-day-old queens and intermediate in reproductive queens. Gene ontology analysis showed that SB/SB queens upregulate reproductive genes faster than SB/Sb queens. For all age classes, genes inside the supergene were overrepresented among the differentially expressed genes. Consistent with the hypothesized greater number of transposons in the Sb supergene, 13 transposon genes were upregulated in SB/Sb queens. Viral genes were also upregulated in SB/Sb mature queens, consistent with the known greater parasite load in colonies headed by SB/Sb queens compared with colonies headed by SB/SB queens. Eighteen differentially expressed genes between reproductive queens were involved in chemical signalling. Our results suggest that many genes in the supergene are involved in regulating social organization and queen phenotypes in fire ants.