Non-synonymous Research Articles

Abstract A37: Interim analysis of CALGB 150607: A pilot study of the mutational & expression status of MET, HGF, EGFR, KRAS, p53, c-CBL, and E-cadherin in resected lung adenocarcinoma specimens

Abstract MET is a receptor tyrosine kinase that plays a critical role in proliferation, angiogenesis, invasion, and metastasis. It has been shown to be dysregulated in a number of malignancies, including non-small cell lung cancer (NSCLC). Despite reports of alterations in MET, and its ligand, HGF, being highly associated with advanced pathological stage and worse outcome in patients with NSCLC, the value of MET as a prognostic biomarker remains unclear. The primary objective of this study is to determine the correlation between MET alterations and expression with stage and overall survival in adenocarcinoma (AC) patients in a large cohort of patients. The secondary objectives are to determine the correlation between overall survival and the following: 1) epithelial mesenchymal transition (EMT), 2) EGFR mutations & expression, 3) KRAS mutations, 4) TP53 mutations, and 5) CBL mutations. In addition, sera levels of circulating MET and HGF will be evaluated in order to determine their potential as prognostic factors. MET, EGFR exons 18–21, TP53 exons 4–10, KRAS exon 2, and CBL exons 2–16 were sequenced using standard PCR and sequencing techniques. Standard immunohistochemistry (IHC) techniques were used to evaluate MET, phosphorylated (pMET Y1003 and Y1230/34/35) p53, HGF, EGFR, and E-cadherin expression. A total of 280 patients will be included in this study, and the interim analysis reported herein evaluated 100 patients. The intensity of cytoplasmic or membranous staining was scored on fourpoint scale: (0, no staining; 1+, weak; 2+, moderate; 3+, strong staining). The extent of staining was scored on a similar scale (0, negative; 1+, 1–10%; 2+, 11–50%; 3+, > 50%). The product of the intensity and extent of staining yielded final scores between 0 and 9. The mean expressions were: MET 3.6 (±0.3); pY1230/34/35 MET 2.0 (±0.2); pY1003MET 4.6 (±0.3); HGF 4.4 (±0.3); EGFR 4.3 (±0.3); TP53 3.7 (±0.3); and E-cadherin 5.5 (±0.3). In ten patient samples, six non-synonymous (NS) mutations were detected in MET (SEMA domain: E168D, M362T, N375S, and Q318K; JM domain: T992I and R970C). In EGFR, the NS mutation L858R was detected in two patients. We detected 12 NS mutations in TP53 (exon 4: E68*, P72R; exon 5: V157F, R175H, I162F, H193Y, Y163D; exon 8: R273L, R273C, V274L, A276F, and G266*). Four NS mutations were detected in exon 2 of KRAS (G12C, G12V, G12D, and G12S). Three mutations were found in the proline-rich region of CBL (L676P, A677S and A678S). ELISAs were utilized to determine soluble MET and HGF levels in pre- and postoperative sera samples. Soluble MET serum levels were significantly increased (p<0.0005) in post-operative samples (1760 ng/ml ± 51.66) compared to pre-operative samples (1585 ng/ml ± 44.61). HGF levels were similar in pre-operative (1008 pg/ml ± 74.8) and post-operative samples (1266 pg/ml ± 175.9). In conclusion, MET and pMET (Y1003) were highly expressed in resected lung AC specimens. NS mutations were detected in all of the oncogenic genes assessed and the functional relevance of these mutations on tumorigenesis remains to be determined. Interestingly, novel MET mutations were detected in key functional domains; the SEMA domain which is critical for dimerization and ligand binding, and the JM domain which is important for MET downregulation via c-CBL. We will correlate these mutational and expression data with clinical outcomes in order to determine the prognostic role of MET.

Differences in mtDNA whole sequence between Tibetan and Han populations suggesting adaptive selection to high altitude

We performed a mitochondrial whole-genome comparison study in 40 Tibetan and 50 Han Chinese. All subjects could be classified into 13 haplogroups pertained to the Macrohaplogroup M and N that pitched different quadrants by principal component analysis. We observed a difference in the M9 haplogroup and identified 18 significant variants by comparing whole sequences between Tibetan and Han populations. Variants in ND2, COX2, tRNA alanine and 12S rRNA were predicted to confer increased protein stability in Tibetans. We compared the base substitutions of nonsynonymous (NS) versus synonymous (S) of 13 protein-encoding genes and found the NS/S values of the ATP6, ATP8, and Cyt b genes were larger (>1) in Tibetans than that in Han population. Our findings provide clues for the existence of adaptive selection for the ATP6, ATP8, Cyt b, ND2, COX2, tRNA alanine and 12S rRNA genes in Tibetans which likely contributed to adaptation to their specific geographic environment, such as high altitude.

Rare versus common variants in pharmacogenetics: SLCO1B1 variation and methotrexate disposition

Methotrexate is used to treat autoimmune diseases and malignancies, including acute lymphoblastic leukemia (ALL). Inter-individual variation in clearance of methotrexate results in heterogeneous systemic exposure, clinical efficacy, and toxicity. In a genome-wide association study of children with ALL, we identified SLCO1B1 as harboring multiple common polymorphisms associated with methotrexate clearance. The extent of influence of rare versus common variants on pharmacogenomic phenotypes remains largely unexplored. We tested the hypothesis that rare variants in SLCO1B1 could affect methotrexate clearance and compared the influence of common versus rare variants in addition to clinical covariates on clearance. From deep resequencing of SLCO1B1 exons in 699 children, we identified 93 SNPs, 15 of which were non-synonymous (NS). Three of these NS SNPs were common, with a minor allele frequency (MAF) >5%, one had low frequency (MAF 1%-5%), and 11 were rare (MAF <1%). NS SNPs (common or rare) predicted to be functionally damaging were more likely to be found among patients with the lowest methotrexate clearance than patients with high clearance. We verified lower function in vitro of four SLCO1B1 haplotypes that were associated with reduced methotrexate clearance. In a multivariate stepwise regression analysis adjusting for other genetic and non-genetic covariates, SLCO1B1 variants accounted for 10.7% of the population variability in clearance. Of that variability, common NS variants accounted for the majority, but rare damaging NS variants constituted 17.8% of SLCO1B1's effects (1.9% of total variation) and had larger effect sizes than common NS variants. Our results show that rare variants are likely to have an important effect on pharmacogenetic phenotypes.

SLCO1B1 Variation and Methotrexate Disposition in Children with Acute Lymphoblastic Leukemia: The Importance of Rare Variants in Pharmacogenetics

Abstract 571Methotrexate is an antifolate chemotherapeutic drug commonly used to treat cancer, including acute lymphoblastic leukemia (ALL). Interindividual variation in clearance of methotrexate results in a vast range of exposure to the drug, which affects its clinical effectiveness and risk of toxicity. In a genome-wide association study of children with ALL, we identified the SLCO1B1 gene, encoding liver transporter OATP1B1, as harboring multiple common polymorphisms associated with methotrexate clearance (Trevino et al. J Clin Oncol, 27:5972-5978, 2009). Because these common polymorphisms could not account for the variation in methotrexate clearance entirely, we hypothesized that there may be rare variants in addition to the common polymorphisms in this gene that affect methotrexate disposition. To test this hypothesis, we sequenced SLCO1B1 exons in 699 pediatric leukemia patients, and compared the effects of common versus rare variants on methotrexate clearance (adjusting for clinical covariates). We identified 93 SNPs, 15 of which were non-synonymous (NS); 11 of the 15 NS SNPs were rare, with minor allele frequencies < 1%. We used four computational programs (SIFT, PMUT, SNPS3D, and Polyphen2) to predict whether the NS SNPs were damaging to the function of the protein. The 7 NS SNPs predicted to be functionally damaging (common or rare) were more likely to be found among patients with the lowest methotrexate clearance than patients with high clearance (p=2.4×10−8), including one SNP that was observed only once among nearly 1400 chromosomes. Four SLCO1B1 haplotypes were associated with reduced methotrexate clearance (p<0.05) and we verified that these haplotypes have lower function with in vitro studies of cellular methotrexate transport. After adjusting for clinical covariates, SLCO1B1 variants accounted for a large portion of the population variability in clearance (p<10−15). Of the variability attributable to SLCO1B1 variants, common NS variants accounted for the majority, but rare damaging NS variants constituted 17.8% of SLCO1B1’s effects on variability in clearance (p<10−3), and had larger effect sizes than common NS variants. Our results show that rare variants are likely to have an important effect on antileukemic agent phenotypes and should not be ignored in clinical pharmacogenetic tests. [Display omitted] Disclosures:Evans:St. Jude Children’s Research Hospital: Employment, Patents & Royalties; NIH NCI: Research Funding; Aldagen: Membership on an entity’s Board of Directors or advisory committees. Relling:Sigma-Tau Pharmaceuticals, Inc: Investigator-initiated research; St. Jude Children’s Research Hospital: Employment, Patents & Royalties; NIH: Research Funding.

Mouse Genome-Wide Association and Systems Genetics Identify Asxl2 As a Regulator of Bone Mineral Density and Osteoclastogenesis

Significant advances have been made in the discovery of genes affecting bone mineral density (BMD); however, our understanding of its genetic basis remains incomplete. In the current study, genome-wide association (GWA) and co-expression network analysis were used in the recently described Hybrid Mouse Diversity Panel (HMDP) to identify and functionally characterize novel BMD genes. In the HMDP, a GWA of total body, spinal, and femoral BMD revealed four significant associations (−log10P>5.39) affecting at least one BMD trait on chromosomes (Chrs.) 7, 11, 12, and 17. The associations implicated a total of 163 genes with each association harboring between 14 and 112 genes. This list was reduced to 26 functional candidates by identifying those genes that were regulated by local eQTL in bone or harbored potentially functional non-synonymous (NS) SNPs. This analysis revealed that the most significant BMD SNP on Chr. 12 was a NS SNP in the additional sex combs like-2 (Asxl2) gene that was predicted to be functional. The involvement of Asxl2 in the regulation of bone mass was confirmed by the observation that Asxl2 knockout mice had reduced BMD. To begin to unravel the mechanism through which Asxl2 influenced BMD, a gene co-expression network was created using cortical bone gene expression microarray data from the HMDP strains. Asxl2 was identified as a member of a co-expression module enriched for genes involved in the differentiation of myeloid cells. In bone, osteoclasts are bone-resorbing cells of myeloid origin, suggesting that Asxl2 may play a role in osteoclast differentiation. In agreement, the knockdown of Asxl2 in bone marrow macrophages impaired their ability to form osteoclasts. This study identifies a new regulator of BMD and osteoclastogenesis and highlights the power of GWA and systems genetics in the mouse for dissecting complex genetic traits.

Epitope variation in the Newcastle disease virus HN gene under antibody immune selective pressure in cell culture

The influence of antibody immune selective pressure on Newcastle disease virus (NDV) HN and F gene mutations was studied in cell cultures. NDV field strain TZ060107 was inoculated into chicken embryo fibroblast cells and continuously passaged with (group A) or without (group B) anti-NDV monospecific serum. Each group contained three independent passage series. HN and F genes were amplified and sequenced for the 10th, 20th, 30th, 40th and 50th generations of each serial passage, and compared with the original strain. The results demonstrated that increased HN gene mutations were observed in group A with the antibody than in group B without the antibody. The nonsynonymous (NS) to synonymous (S) mutations ratio was 6 for group A, significantly higher than 3.4 in group B. In group A with the antibody, there were five stable NS mutations in HN gene, three of which (related to aa#353, 521 and 568) were related to known epitopes. There were two stable NS mutations in F gene in group A, but no stable NS mutations in group B. The NS/S ratios of F gene were less than 2.5 for both groups A and B. Our results suggested that the antibody strongly influenced HN gene mutations, while the F gene was less influenced by the same antibody.

Molecular Characterization of the NPC1L1 Variants Identified from Cholesterol Low Absorbers

Niemann-Pick C1-like 1 (NPC1L1) is an essential protein for dietary cholesterol absorption. Nonsynonymous (NS) variants of NPC1L1 in humans have been suggested to associate with cholesterol absorption variations. However, information concerning the characteristics and mechanism of these variants in cholesterol uptake is limited. In this study, we analyzed the cholesterol uptake ability of the 19 reported NS variants of NPC1L1 identified from cholesterol low absorbers. Among these variants, L110F, R306C, A395V, G402S, T413M, R693C, R1214H, and R1268H could partially mediate cellular cholesterol uptake and were categorized as partially dysfunctional variants. The other 11 variants including T61M, N132S, D398G, R417W, G434R, T499M, S620C, I647N, G672R, S881L, and R1108W could barely facilitate cholesterol uptake, and were classified into the severely dysfunctional group. The partially dysfunctional variants showed mild defects in one or multiple aspects of cholesterol-regulated recycling, subcellular localization, glycosylation, and protein stability. The severely dysfunctional ones displayed remarkable defects in all these aspects and were rapidly degraded through the ER-associated degradation (ERAD) pathway. In vivo analyses using adenovirus-mediated expression in mouse liver confirmed that the S881L variant failed to localize to liver canalicular membrane, and the mice showed defects in biliary cholesterol re-absorption, while the G402S variant appeared to be similar to wild-type NPC1L1 in mouse liver. This study suggests that the dysfunction of the 19 variants on cholesterol absorption is due to the impairment of recycling, subcellular localization, glycosylation, or stability of NPC1L1.

Comparative Gene Evolution in Haemosporidian (Apicomplexa) Parasites of Birds and Mammals

Haemosporidian parasites of birds and mammals reproduce asexually inside nucleated and nonnucleated host erythrocytes, respectively. Because of these different parasite environments and because bird parasites are paraphyletic, we evaluated whether patterns of parasite molecular evolution differ between host groups. We compared two mitochondrial (mt) genes and one apicoplast gene across mammal Plasmodium, bird Plasmodium, and bird Parahaemoproteus. Using a molecular phylogenetic approach, we show that the parasite mt cytochrome b (cyt b), mt cytochrome oxidase I (COI), and the apicoplast caseinolytic protease C (ClpC) exhibit similar levels of sequence divergence, yet each gene tree presents a strikingly different pattern of internal versus terminal branch lengths. In cyt b, the ratio of nonsynonymous (NS)-to-synonymous substitutions (d(N)/d(S)) is markedly elevated along the internal branch linking mammalian and avian parasites despite the sister relationship between mammal and bird Plasmodium. This is not the case for either COI or ClpC. When NS substitutions are excluded from the parasite cyt b alignment, the resulting phylogenetic tree resembles that of COI (both with and without NS substitutions). The high d(N)/d(S) ratio in the cyt b branch separating avian and mammalian parasites and a mammal-parasite codon bias suggest that adaptive evolution has distinguished mammal and bird parasites.

Identification and Characterization of Human Genes Targeted by Natural Selection

Abstract The human genome has evolved as a consequence of evolutionary forces, such as natural selection. In this study, we investigated natural selection on the human genes by comparing the numbers of nonsynonymous (NS) and synonymous (S) mutations in individual genes. We initially collected all coding SNP data of all human genes from the public dbSNP. Among the human genes, we selected 3 different selection groups of genes: pos-itively selected genes (NS/S≥3), negatively selected genes (NS/S≤1/3) and neutral selection genes (0.9 ＜NS/S＜1.1). We characterized human genes targeted by natural selection. Negatively selected human genes were markedly associated with disease occurrence, but not positively selected genes. Interestingly, positively se-lected genes displayed an increase in potentially delete-rious nonsynonymous SNPs with an increased fre-quency of tryptophan and tyrosine residues, suggesting a correlation with protective effects against human disease. Furthermore, our nonsynonymous/synonymous ratio data imply that specific human genes, such as ALMS1 and SPTBN5 genes, are differentially selected among distinct populations. We confirmed that in-ferences of natural selection using the NS/S ratio can be used extensively to identify functional genes selected during the evolutionary adaptation process.Keywords: single nucleotide polymorphism (SNP), natu-ral selection, disease genes, ethnicity

Rare loss-of-function mutations in ANGPTL family members contribute to plasma triglyceride levels in humans

The relative activity of lipoprotein lipase (LPL) in different tissues controls the partitioning of lipoprotein-derived fatty acids between sites of fat storage (adipose tissue) and oxidation (heart and skeletal muscle). Here we used a reverse genetic strategy to test the hypothesis that 4 angiopoietin-like proteins (ANGPTL3, -4, -5, and -6) play key roles in triglyceride (TG) metabolism in humans. We re-sequenced the coding regions of the genes encoding these proteins and identified multiple rare nonsynonymous (NS) sequence variations that were associated with low plasma TG levels but not with other metabolic phenotypes. Functional studies revealed that all mutant alleles of ANGPTL3 and ANGPTL4 that were associated with low plasma TG levels interfered either with the synthesis or secretion of the protein or with the ability of the ANGPTL protein to inhibit LPL. A total of 1% of the Dallas Heart Study population and 4% of those participants with a plasma TG in the lowest quartile had a rare loss-of-function mutation in ANGPTL3, ANGPTL4, or ANGPTL5. Thus, ANGPTL3, ANGPTL4, and ANGPTL5, but not ANGPTL6, play nonredundant roles in TG metabolism, and multiple alleles at these loci cumulatively contribute to variability in plasma TG levels in humans.

Sporadic Bilateral Optic Neuropathy in Children: The Role of Mitochondrial Abnormalities

To evaluate a group of patients with isolated, early-onset, bilateral optic neuropathy for genetic and biochemical evidence of mitochondrial diseases. This case-control study involved 21 patients, 159 control subjects for mitochondrial (mt)DNA sequencing, and 40 control subjects for relative mtDNA content. Patients were identified who had had decreased vision since childhood due to bilateral optic neuropathy characterized by central visual loss with no other major neurologic or ocular abnormality and no clinical evidence of a mitochondrial syndrome. Clinical examination, electroretinograms, and neuroimaging were performed; the entire mtDNA coding region was sequenced in leukocytes of all patients; relative mtDNA content was assessed; and OPA1 and OPA3 nuclear genes associated with dominant and recessive optic atrophy, respectively, were sequenced. Main outcome measures were clinical description, nonsynonymous (NS) mtDNA nucleotide changes, relative mtDNA content, and OPA1 and OPA3 nucleotide changes. Twenty-one unrelated patients (16 male and 5 female; mean age at first examination 13.6 years) had bilateral moderate, relatively symmetric optic neuropathies and normal neurologic examinations other than strabismus in 11 and congenital nystagmus in 9. Four patients had optic nerve hypoplasia. One patient had the nt 11778 primary Leber hereditary optic neuropathy (LHON) mutation, and three others had mtDNA nucleotide changes predicted to be pathologic. The entire group had a small increase (6.7%) in relative mtDNA content of indeterminate statistical significance. No patient had a polymorphism or mutation of OPA1 or OPA3. A minority of these young patients with sporadic bilateral optic neuropathy had abnormalities of the mitochondrial parameters evaluated. This bilateral optic neuropathy may be due to other genetic, epigenetic, or environmental injury to the optic nerve or to mitochondrial defects not studied.

Distributions of single nucleotide polymorphisms in differential chromosome segments of congenic resistant strains that define minor histocompatibility antigens

Minor histocompatibility antigens (MiHAs) stimulate the rejection of allografts when donors and recipients are matched at the major histocompatibility complex (MHC). The majority of identified autosomal MiHAs were generated by non-synonymous (NS) substitutions that alter MHC class I-binding peptides. The mosaic distribution of single nucleotide polymorphisms (SNPs) that distinguish inbred mouse strains led us to hypothesize that MiHA genes defined by congenic strains on C57BL/6 and C57BL/10 backgrounds map to chromosomal regions with relatively high numbers of NS SNPs that distinguish C57 strains from other common inbred strains. To test this hypothesis, we mapped the ends of differential chromosome segments of congenic strains, which define 12 MiHAs, relative to microsatellites and SNPs. The lengths of differential segments ranged from 9.7 to 105.9 Mbp in congenic strains where no attempts were made to select recombinants within these segments. There was no apparent correlation between differential segment length and number of backcrosses, suggesting that factors other than the number of opportunities for recombination affected the differential segment lengths in these congenics. These differential segments included higher numbers of NS SNPs that distinguish C57BL/6J from A/J, DBA/2J, and 129S1/J than would be predicted if these SNPs were uniformly distributed along the chromosomes. The most extreme case was the H8 congenic that included 74% of the SNPs on chromosome 14 within its 9.7-11.1 Mbp differential segment. These results point toward a direct relationship between the level of genomic divergence, as indicated by numbers of NS SNPs, and numbers of MiHAs that collectively determine the magnitude of allograft rejection.

Genetic diversity and function in the human cytosolic sulfotransferases

Amino-acid substitutions, which result from common nonsynonymous (NS) polymorphisms, may dramatically alter the function of the encoded protein. Gaining insight into how these substitutions alter function is a step toward acquiring predictability. In this study, we incorporated gene resequencing, functional genomics, amino-acid characterization and crystal structure analysis for the cytosolic sulfotransferases (SULTs) to attempt to gain predictability regarding the function of variant allozymes. Previously, four SULT genes were resequenced in 118 DNA samples. With additional resequencing of the remaining eight SULT family members in the same DNA samples, a total of 217 polymorphisms were revealed. Of 64 polymorphisms identified within 8785 bp of coding regions from SULT genes examined, 25 were synonymous and 39 were NS. Overall, the proportion of synonymous changes was greater than expected from a random distribution of mutations, suggesting the presence of a selective pressure against amino-acid substitutions. Functional data for common variants of five SULT genes have been previously published. These data, together with the SULT1A1 variant allozyme data presented in this paper, showed that the major mechanism by which amino acid changes altered function in a transient expression system was through decreases in immunoreactive protein rather than changes in enzyme kinetics. Additional insight with regard to mechanisms by which NS single nucleotide polymorphisms alter function was sought by analysis of evolutionary conservation, physicochemical properties of the amino-acid substitutions and crystal structure analysis. Neither individual amino-acid characteristics nor structural models were able to accurately and reliably predict the function of variant allozymes. These results suggest that common amino-acid substitutions may not dramatically alter the protein structure, but affect interactions with the cellular environment that are currently not well understood.

Multiple rare variants in NPC1L1 associated with reduced sterol absorption and plasma low-density lipoprotein levels

An approach to understand quantitative traits was recently proposed based on the finding that nonsynonymous (NS) sequence variants in certain genes are preferentially enriched at one extreme of the population distribution. The NS variants, although individually rare, are cumulatively frequent and influence quantitative traits, such as plasma lipoprotein levels. Here, we use the NS variant technique to demonstrate that genetic variation in NPC1L1 contributes to variability in cholesterol absorption and plasma levels of low-density lipoproteins (LDLs). The ratio of plasma campesterol (a plant sterol) to lathosterol (a cholesterol precursor) was used to estimate relative cholesterol absorption in a population-based study. Nonsynonymous sequence variations in NPC1L1 were five times more common in low absorbers (n = 26 of 256) than in high absorbers (n = 5 of 256) (P < 0.001). The rare variants identified in low absorbers were found in 6% of 1,832 African-Americans and were associated with lower plasma levels of LDL cholesterol (LDL-C) (96 +/- 36 mg/dl vs. 105 +/- 36 mg/dl; P = 0.005). These data, together with prior findings, reveal a genetic architecture for LDL-C levels that does not conform to current models for quantitative traits and indicate that a significant fraction of genetic variance in LDL-C is due to multiple alleles with modest effects that are present at low frequencies in the population.

Human Immunodeficiency Virus Mutations during the First Month of Infection Are Preferentially Found in Known Cytotoxic T-Lymphocyte Epitopes

The full protein coding region of human immunodeficiency virus (HIV) genomes were sequenced using plasma collected from nine African-Americans prior to seroconversion and 7 to 28 days later. HIV mutations emerged in seven of these subjects at a genomewide rate of 2% per year. The location of nonsynonymous (NS) HIV mutations within these subjects was compared to their potential HLA-A and B types restricted CTL epitopes reported in the Los Alamos National Laboratory HIV immunology database. A statistically significant (P < 0.005) number of the early NS mutations (13.5%) were found within previously reported CTL epitopes. A virus sequencing and reported CTL epitopes database analysis therefore support a model where a significant proportion of very early nonsynonymous HIV mutations are selected by CTL.

A large-scale study of the random variability of a coding sequence: a study on the CFTR gene

Coding single nucleotide substitutions (cSNSs) have been studied on hundreds of genes using small samples (n(g) approximately 100-150 genes). In the present investigation, a large random European population sample (average n(g) approximately 1500) was studied for a single gene, the CFTR (Cystic Fibrosis Transmembrane conductance Regulator). The nonsynonymous (NS) substitutions exhibited, in accordance with previous reports, a mean probability of being polymorphic (q > 0.005), much lower than that of the synonymous (S) substitutions, but they showed a similar rate of subpolymorphic (q < 0.005) variability. This indicates that, in autosomal genes that may have harmful recessive alleles (nonduplicated genes with important functions), genetic drift overwhelms selection in the subpolymorphic range of variability, making disadvantageous alleles behave as neutral. These results imply that the majority of the subpolymorphic nonsynonymous alleles of these genes are selectively negative or even pathogenic.

HVR-1 quasispecies modifications occur early and are correlated to initial but not sustained response in HCV-infected patients treated with pegylated- or standard-interferon and ribavirin

Background/Aims HVR-1 quasispecies composition and evolution were investigated in patients chronically infected with genotype 1b HCV, treated with PEG-IFN α2b or STD-IFN α2b plus RBV. Methods HVR-1 heterogeneity was assessed by calculating nucleotidic complexity, diversity, synonymous (S) and non-synonymous (NS) substitutions at baseline, after 4 weeks of therapy ( T1) and at follow-up ( T18). Evolution of viral quasispecies was analysed by constructing phylogenetic trees. Results No correlation of baseline viremia with heterogeneity was observed. Nucleotidic complexity was lower in patients showing early virological response, and tended to be inversely correlated to viral load decline at 4 weeks of treatment. In the majority of SR, profound changes of quasispecies composition occurred during 4 weeks of treatment, while in NR virtually no major changes of pre-therapy variants were observed. Relapse showed both patterns of quasispecies evolution. Virus quasispecies after follow-up was similar to that found at T1 in both Relapsers and NR patients. Conclusions Baseline parameters of HVR-1 heterogeneity seem to be involved in the early response to treatment, and early response is associated with profound variations in the HVR-1 quasispecies. Viral quasispecies surviving early therapeutic pressure are most likely able to give rise to either virus rebound or persistence at T18.

Intra- and interspecific variation of the CCR5 gene in higher primates.

We have evaluated the molecular evolution of the chemokine receptor CCR5 in primates. The chemokine receptor CCR5 serves as a major co-receptor for human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infection. Knowledge of evolution of the CCR5 molecule and selection on the CCR5 gene may shed light on its functional role. The comparison of differences between intraspecific polymorphisms and interspecific fixed substitutions provides useful information regarding modes of selection during the course of evolution. There is marked polymorphism in the CCR5 gene sequence within different primate species, whereas sequence divergence between different species is small. By using contingency tests, we compared synonymous (SS) and nonsynonymous (NS) CCR5 mutations occurring within and between a broad range of primates. Our results demonstrate that CCR5 evolution did not follow expectations of strict neutrality at the level of the whole gene. The proportion of NS to SS at the intraspecific level was significantly higher than that observed at the interspecific level. These results suggest that most CCR5 NS polymorphisms are slightly deleterious. However, at domains more closely correlated with its known biological functions, there was no obvious evidence to support deviation from neutrality.