Total Number Of Mutations Research Articles

Transmitted Antiretroviral Drug Resistance in Individuals with Newly Diagnosed HIV Infection: South Carolina 2005–2009

The transmission of drug-resistant human immunodeficiency virus 1 (HIV-1) has important implications for the antiretroviral management of newly diagnosed individuals, increasing the risk of suboptimal treatment outcomes. The study objective was to characterize rates and factors associated with transmitted drug-resistant HIV-1 infection among newly diagnosed South Carolina (SC) residents. This study utilized surveillance genotypic data from antiretroviral therapy (ART)-naïve individuals newly diagnosed with HIV-1 infection from June 2005 through December 2009. Multivariable negative binomial regression was used to model the association between the presence of major mutations and sociodemographic characteristics. Of the 1,277 study participants, 14.4% (184/1,277) had HIV-1 variants with major antiretroviral drug mutations. Of these individuals, 126 had non-nucleoside reverse transcriptase inhibitor-associated mutations (NNRTI), 54 had nucleos(t)ide reverse transcriptase inhibitor-associated mutations (NRTI), 37 had protease inhibitor-associated mutations (PI). Nineteen (10.3%) individuals had dual class-associated mutations (NNRTI and PI in seven, NNRTI and NRTI in seven, and NRTI and PI in five individuals), and seven (3.8%) individuals had triple drug class-associated mutations (PI, NNRTI, and NRTI). The multivariable negative binomial regression models indicated that age at HIV diagnosis had a significant negative association with total number of mutations (rate ratio [RR] 0.88, 95% confidence interval [CI] 0.80-0.96, P value=0.005) and total number of reverse transcriptase (RT) mutations (RR 0.88, 95% CI 0.80-0.97, P value=0.006) present. Prevalence of transmitted drug resistance is consistently high among newly diagnosed HIV-infected individuals in SC. It is important to continue genotypic surveillance to facilitate effective HIV treatment and empiric post-exposure prophylaxis regimens.

Molecular approach to evaluate the genotoxicity of glyphosate (roundup) using mosquito genome

Glyphosate, an active ingredient in Roundup is a broad spectrum, systemic and non -selective herbicide which is commonly used for eliminating weeds in agriculture and forest landscapes. The present studies deal with the evaluation of the genotoxic potential of Glyphosate with two different dose concentration of LD20 and LD40 on a mosquito Culex quinquefasciatus taken as an experimental model. For this, polymerase chain reaction technique (PCR) was used for detecting DNA damage by amplifying ribosomal DNA internal transcribed spacer 2 (ITS 2) region. The amplified products were sequenced and the results of treated and non-treated controls were compared by using Clustal W software programme. The results were studied in the form of transitions, transversions, deletions and additions of bases. The DNA band amplified from control stocks consisted of 440 bases while thosefrom LD20 and LD40 treated individuals were comprised of 423 and 468 bases respectively. The total number of mutations caused in LD20 treated stock was 205 out of which 68 were transitions, 90 transversions, 32 deletions and 15 additions. In case of LD40 treated individuals, as many as 221 bases had suffered mutations, out of which 66 were transitions, 90 transversions , 12 deletions and 41 additions. In both the cases the rate of transversions was higher than transitions. From these results it was evident that glyphosate has a potential to promote gene mutations in the individuals exposed to its semilethal doses.

The mutational archive in proviral DNA does not change during 24 months of continuous or intermittent highly active antiretroviral therapy

The aim of the study was to determine the modifications of the mutational archive in proviral HIV-1 DNA occurring during 24 months of intermittent or continuous highly active antiretroviral therapy (HAART). The study population included subjects enrolled in the Istituto Superiore di Sanità Pulsed Antiretroviral Therapy (ISS PART) clinical trial. All of these patients were on first-line HAART and had plasma HIV-1 RNA below 50 HIV-1 RNA copies/mL. A genotypic resistance test was performed on HIV-1 DNA extracted from peripheral blood mononuclear cells (PBMC) at baseline and after 24 months of follow-up. Resistance-associated mutations (RAMs) were defined according to the International AIDS Society (IAS) USA classification. Sixty-nine subjects were included in the study [36 enrolled in arm A of the ISS PART (continuous HAART) and 33 enrolled in arm B (intermittent HAART)]. No major modifications of the mutational archive were found in either group after 24 months of follow-up, in terms of both the proportion of subjects with mutations and the total number of mutations. In this patient population, the mutational archive in HIV-1 DNA extracted from PBMC was stable for 24 months, irrespective of HAART modality, whether continuous or intermittent.

Accuracy estimation of foamy virus genome copying

BackgroundFoamy viruses (FVs) are the most genetically stable viruses of the retrovirus family. This is in contrast to the in vitro error rate found for recombinant FV reverse transcriptase (RT). To investigate the accuracy of FV genome copying in vivo we analyzed the occurrence of mutations in HEK 293T cell culture after a single round of reverse transcription using a replication-deficient vector system. Furthermore, the frequency of FV recombination by template switching (TS) and the cross-packaging ability of different FV strains were analyzed.ResultsWe initially sequenced 90,000 nucleotides and detected 39 mutations, corresponding to an in vivo error rate of approximately 4 × 10-4 per site per replication cycle. Surprisingly, all mutations were transitions from G to A, suggesting that APOBEC3 activity is the driving force for the majority of mutations detected in our experimental system. In line with this, we detected a late but significant APOBEC3G and 3F mRNA by quantitative PCR in the cells. We then analyzed 170,000 additional nucleotides from experiments in which we co-transfected the APOBEC3-interfering foamy viral bet gene and observed a significant 50% drop in G to A mutations, indicating that APOBEC activity indeed contributes substantially to the foamy viral replication error rate in vivo. However, even in the presence of Bet, 35 out of 37 substitutions were G to A, suggesting that residual APOBEC activity accounted for most of the observed mutations. If we subtract these APOBEC-like mutations from the total number of mutations, we calculate a maximal intrinsic in vivo error rate of 1.1 × 10-5 per site per replication. In addition to the point mutations, we detected one 49 bp deletion within the analyzed 260000 nucleotides.Analysis of the recombination frequency of FV vector genomes revealed a 27% probability for a template switching (TS) event within a 1 kilobase (kb) region. This corresponds to a 98% probability that FVs undergo at least one additional TS event per replication cycle. We also show that a given FV particle is able to cross-transfer a heterologous FV genome, although at reduced efficiency than the homologous vector.ConclusionOur results indicate that the copying of the FV genome is more accurate than previously thought. On the other hand recombination among FV genomes appears to be a frequent event.

Improved Prognosis of Chronic Lymphocytic Leukemia (CLL) Patients with Increased IgVH Mutations May Reflect Greater Alteration of the B-Cell Receptor (BCR) Binding Site

BACKGROUND: IgVH mutation status is a well accepted prognostic marker in CLL. Typically >98% sequence homology to the most similar germline gene is considered unmutated, and ≤98% is considered mutated. The 98% cutoff was originally chosen to account for undefined IgVH polymorphisms that could be responsible for DNA sequence disparity. Prior data showed that mutated patients have a more benign course of disease vs. unmutated patients. Although it has been presumed that this is due to a structural change in the BCR, specific consideration of the nature of the mutations (silent or replacement) and the relation to clinical course has not been made. We recently showed that IgVH somatic mutation correlates to disease course in a continuous fashion; prognosis improves as the number of mutations increases. Therefore, the present study was designed to determine if replacement mutations were more important than silent mutations or if the “mutation process” (reflected as the total number of mutations, irrespective of their structural impact) was responsible for improved survivalMETHODS: We sought to determine if further characterization of mutations (replacement vs. silent) and location thereof (framework (FR) v. complementarity determining region (CDR)) changed with increasing number of mutations. We hypothesized the ratio of replacement-to-silent (R:S) mutations would be greater in those with better prognosis because such changes could significantly alter the structure of the antigen-binding site and that this would be more pronounced in the CDRs. Survival analysis (Kaplan-Meier, logrank test (overall and adjusted pairwise), Cox regression) was used to analyze both outcomes of time to treatment (TTT) and survival (OS). Quartiles of numbers of mutations were used to determine how outcome varied accordingly. The range of mutation in each quartile divided as follows: 1–9; 10–15; 11–20; and 21–39. We examined the ratio of R:S mutations to determine whether the ratios varied according to quartile (Mann-Whitney test).RESULTS: 353 patients were analyzed. 89 of 334 (27%) had received treatment and 53 of 353 (15%) had died. The lower quartile of mutations (1–9) had a significantly shorter TTT and OS than the three higher quartiles (P< 0.0006, P<0.0014, respectively, logrank test); the three upper quartiles did not differ statistically from one another. Among mutations in the CDR, there was a significant difference in the R:S ratio between the lower quartile of mutation number and the combined 3 upper quartiles. (Mean R:S = 3.25 in Q1, 5.64 in Q2–Q4, P<0.0008). Among mutations in the FR, R:S did not differ significantly between quartile groups (Mean R:S = 2.44 in Q1, 2.19 in Q2–Q4). Among all mutations (FR and CDR), R:S ratio did not differ significantly between quartile groups. (Mean R:S = 2.81 in Q1, 2.55 in Q2–Q4).CONCLUSION: The association of better prognosis with more IgVH mutations may reflect the higher ratio of R:S mutations in the CDR. These data have implications as to the role of the CLL B-cell receptor (BCR) and its specificity in clinical outcome. Assuming that in vivo BCR stimulation causes CLL cell proliferation, increased replacement mutations in the CDRs would lead to more binding site structural or conformational change and thereby narrow antigen-binding range. This would limit the diversity of antigens capable of delivering a stimulatory signal. That no increase in R:S was found in the FRs is consistent with these regions' function as a scaffolding to the BCR, the structural alteration of which could render the BCR ineffective.Patients divided into quartiles according to mutation numberQuartile 1Quartiles 2-4p-valueNumber of Mutations1–910–36Hazard Ratio: TUT2.37<0.0001Hazard Ratio: OS2.900.0003Regionn PtsMean R:S ration PtsMean R:S ratioCDR + FR942.812592.55not signifFR802.442592.18not signifCDR663.252585.640.0008R:S in CDR and FR could not be calculated for those patients with no mutations in the corresponding region.

Does sex induce a phase transition?

We discovered a dynamic phase transition induced by sexual reproduction. The dynamics is a pure Darwinian rule with both fundamental ingredients to drive evolution: 1) random mutations and crossings which act in the sense of increasing the entropy (or diversity); and 2) selection which acts in the opposite sense by limiting the entropy explosion. Selection wins this competition if mutations performed at birth are few enough. By slowly increasing the average number m of mutations, however, the population suddenly undergoes a mutational degradation precisely at a transition point mc. Above this point, the bad alleles spread over the genetic pool of the population, overcoming the selection pressure. Individuals become selectively alike, and evolution stops. Only below this point, m < mc, evolutionary life is possible. The finite-size-scaling behaviour of this transition is exhibited for large enough lengths L. One important and surprising observation is the L-independence of the transition curves, for large L. They are also independent on the population size. Another is that mc is near unity, i.e. life cannot be stable with much more than one mutation per diploid genome, independent of the chromosome length, in agreement with reality. One possible consequence is that an eventual evolutionary jump towards larger L enabling the storage of more genetic information would demand an improved DNA copying machinery in order to keep the same total number of mutations per offspring.

852 A MUTATIONAL ANALYSIS OF THE INTERNAL RIBOSOME ENTRY SITE IN HEPATITIS C VIRUS IN PATIENTS TREATED WITH PEGYLATED INTERFERON WITH RIBAVIRIN

Background and Aims: It has recently been reported that the internal ribosome entry site (IRES) is closely associated with the replication of the hepatitis C virus (HCV) RNA. On the other hand, the HCVRNA kinetics is an important predictive factor for the outcome of antiviral therapy. To investigate the potential mechanisms of resistance, especially regarding the HCVRNA kinetics, we analyzed the mutation of the IRES in chronic hepatitis C (CH-C) patients treated by pegylated interferon (Peg-IFN) with ribavirin (RBV). Methods: 31 CH-C patients demonstrating genotype 1b with a high viral load (more than 100 KIU/ml) were studied. They were divided into two groups. The 12w (+) group included 15 patients, whose HCVRNA were positive at 12 weeks after the start of therapy. In contrast, the 12w (−) group included 16 patients whose HCVRNA were negative at 12 weeks. HCVRNA was extracted from the serum just before therapy in both groups, and the IRES region was sequenced. Three clones were analyzed in each patient. Five patients in the 12w (+) group were further analyzed in the serum at 12 weeks. Results: At week 0, the total number of mutations from each clone in the IRES (domain II/III) in the 12w (+) group was 19/9, in contrast to 14/35 in the 12w (−) group (P = 0.0008). Namely, in the 12w (+) group, most of the mutations were observed in domain II. On the other hand, in the 12w (−) group, the mutations were concentrated in domain III. No other clinical factors were statistically significant. Furthermore, in comparison of the findings at week 0 and week 12 in 5 patients within the 12w (+) group, the number of mutations in domain II/III was 9/4 in week 0, in contrast to 8/1 in week 12. Namely, the mutations of domain III decreased. Conclusions: The mutations of the IRES were found to be related to the HCVRNA kinetics, in particular, mutations in domain II seem to be associated with the resistance of Peg-IFN with RBV therapy.

Observational Study on HIV-Infected Subjects Failing HAART Receiving Tenofovir Plus Didanosine as NRTI Backbone

We evaluated the efficacy of tenofovir (TDF) - and didanosine (ddI)-containing backbones in HIV-infected experienced subjects. We included in the study 245 subjects who started a TDF/ddI-containing HAART with HIV-RNA > 3 log(10) cp/ml and an available genotypic resistance test at baseline. At baseline, median CD4 counts and HIV-RNA were 278 cell/mmc and 4.32 log(10) cp/ml, respectively. Seventy-four subjects (30.2%) discontinued TDF and/or ddI, 23 of them for drug-related toxicities or intolerance. One-hundred and twenty-six (51.4%) subjects achieved virologic success (HIV-RNA < 50 copies/ml in two consecutive determinations) in a median time of 6.1 months; higher HIV-RNA levels (HR: 0.66, 95% CI: 0.54- 0.79, p < 0.001 for each additional log(10) copies/ml), and the total number of mutations either for PI and NNRTI at baseline (HR: 0.87, 95% CI: 0.81-0.92, p < 0.001 for each additional mutation) were both predictors of virologic success. M184V was marginally associated with virologic success (HR: 1.34, 95% CI: 0.94-1.90, p = 0.10 vs no M184V), whilst the number of TAMs was not associated. One-hundred-thirty-three (54.3%) subjects achieved immunologic success (increase of > or = 100 cells/mm(3) from baseline) in a median time of 7.5 months; immunologic success was associated with HIV-RNA levels at baseline (HR: 0.91, 95% CI: 0.79-0.98, p = 0.04 for each additional log(10) copies/ml), the total number of mutations either for PI or NNRTI (HR: 0.91, 95% CI: 0.85-0.98, p = 0.01 for each additional mutation) and CD4 count at baseline (HR: 1.11, 95% CI: 1.00-1.23, p = 0.05 for each additional 100 cells/mm(3)). Results obtained by the on-treatment analyses were comparable. In our study, HAART containing TDF/ddI seem associated with a virologic and immunologic response, when such regimens are chosen according to a genotypic resistance test.

Molecular and epidemiological characterization of rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis in Kyrgyz Republic

The nature and frequency of mutations in the rpoB gene of M. tuberculosis (MBT) vary considerably in various geographical locations. There is no information on the prevalence of specific mutations in the rpoB gene of MBT isolated from patients in Kyrgyz Republic. In this work, we analyzed a distribution of the rpoB gene mutations in Kyrgyz Republic. A total of 380 rifampicin-sensitive and 225 rifampicin-resistant MBT cultures were analyzed to identify and to characterize mutations in the rpoB gene using a biological microchip assay. The biochip test determined 18 different mutation types in 8 codons of the rifampicin-resistant samples. The majority of mutations (180 of 225, or 80 %) were in the codons 531 and 526, mainly in the codon 531 (137 of 225, 60.8 %). The Ser531&gt;Leu mutation (134 of 225, 59.4 %) was by far the most common. Another group of mutations were in the codon 526 (43 of 225, 19.1 %). Five different types of mutations were found in the codon 526 which were: His526®Tyr (4.9 %), His 526®Asp (4.9 %), His526®Arg (4.0 %), His526®Leu (3.5 %), and His526®Pro (1.8 %). The third group of common mutations were Leu511®Pro (6.3 %) and Asp516®Tyr (4.4 %). Other mutations found in the codons 533, 522, 513, and 512 were less frequent and had a very low rate comprising about 1.8 % of the total mutation number among 225 rifampicinresistant samples.

Evolution and Differentiation of MSHR Gene in Different Species

Coat color offers some prospects for evolutionary studies due to its large amount of presumably adaptive coat color variation and conserved genetic mechanisms of generating different coat colors in different species. Melanocyte-stimulating hormone receptor (MSHR) gene is responsible for intraspecific and interspecific color variation in mammals and birds. A total number of 206 MSHR gene sequences belonging to 84 species, 58 genera, and 20 families were analyzed to investigate its evolution and differentiation in different species. Most of the species have 954 bp and stop codon TGA. Species in Callithrix and Callimico have a stop codon mutation from TGA to TGG and elongate 81 bp with TAG as stop codon. Species in Phasianidae, Fringillidae, and Lemuridae also use TAG as stop codon. The Sus scrofa had an insertion of AACCAGACC encoding Asn-Gln-Thr from 85 to 93 bp. In Bovidae, a brown strain of cow with 966 bp due to the 12-bp duplication of GGCATTGCCCGG from 670 to 681 bp encoding for Gly-Ile-Ala-Arg was found. Teiidae has the smallest number of total mutations (6), silent mutations (3), nonsynonymous mutations (3), average number of nucleotide differences (1.519), synonymous nucleotide diversity (pi(s) = 0.0030), and nonsynonymous nucleotide diversity (pi(a) = 0.0029), and Hominidae, Lemuridae, Canidae, and Teiidae have higher ratio of pi(a)/pi(s) (0.537-0.973). The reconstructed phylogenetic tree of MSHR gene of families is basically consistent with the taxonomy of National Center for Biotechnology Information.

Multiple Thrombophilic Gene Mutations Rather than Specific Gene Mutations are Risk Factors for Recurrent Miscarriage

Recurrent miscarriage is a heterogeneous condition. While the role of acquired thrombophilia has been accepted as an etiology of recurrent miscarriage, the contribution of specific inherited thrombophilic genes to this disorder has remained controversial. We compared the prevalence of 10 thrombophilic gene mutations among women with a history of recurrent miscarriages and fertile control women. A total of 150 women with a history of two or more recurrent pregnancy losses and 20 fertile control women with no history of pregnancy losses had buccal swabs taken for DNA analyses of 10 gene mutations [factor V G1691A, factor V H1299R (R2), factor V Y1702C, factor II prothrombin G20210A, factor XIII V34L, beta-fibrinogen -455G>A, PAI-1 4G/5G, HPA1 a/b (L33P), MTHFR C677T, MTHFR A1298C]. The prevalence of these mutations was compared between women experiencing recurrent miscarriage and controls. No differences in the frequency of specific gene mutations were detected when women with recurrent miscarriage were compared with control women. However, the prevalence of homozygous mutations and total gene mutations among patients with recurrent miscarriage was significantly higher than among controls. Homozygous mutations were found in 59% of women with a history of recurrent pregnancy loss contrasted to 10% of control women. More than three gene mutations among the 10 genes studied were observed in 68% of women with recurrent miscarriage and 21% of controls. Inherited thrombophilias are associated with recurrent miscarriage. This association is manifest by total number of mutations rather than specific genes involved.

Multiple thrombophilic gene mutations are risk factors for implantation failure

While the role of inherited thrombophilia has been accepted as a cause of recurrent late pregnancy complications, the contribution of mutated thrombophilic genes to implantation failure has not been studied. Proteins involved in fibrinolysis are necessary for trophoblast invasion into the endometrium. This study compared the prevalence of 10 thrombophilic gene mutations among 42 women with a history of recurrent implantation failure after IVF–embryo transfer with 20 fertile control women. Buccal swabs were taken from all of the women for DNA analyses. Women with a history of implantation failure after IVF–embryo transfer displayed a higher prevalence of PAI-1 4G/5G mutations than controls (P = 0.007). No differences in the frequency of the other specific gene mutations were detected. However, the prevalence of total gene mutations among patients with implantation failure was significantly higher than among controls. More than three gene mutations among the 10 genes studied were observed in 74% of women with implantation failure and 20% of controls (P = 0.0004). It is concluded that inherited thrombophilias are associated with implantation failure. This association is manifest by total number of mutations as well as with PAI-1 mutations.

Genomic characterization reveals a simple histone H4 acetylation code

The histone code hypothesis holds that covalent posttranslational modifications of histone tails are interpreted by the cell to yield a rich combinatorial transcriptional output. This hypothesis has been the subject of active debate in the literature. Here, we investigated the combinatorial complexity of the acetylation code at the four lysine residues of the histone H4 tail in budding yeast. We constructed yeast strains carrying all 15 possible combinations of mutations among lysines 5, 8, 12, and 16 to arginine in the histone H4 tail, mimicking positively charged, unacetylated lysine states, and characterized the resulting genome-wide changes in gene expression by using DNA microarrays. Only the lysine 16 mutation had specific transcriptional consequences independent of the mutational state of the other lysines (affecting approximately 100 genes). In contrast, for lysines 5, 8, and 12, expression changes were due to nonspecific, cumulative effects seen as increased transcription correlating with an increase in the total number of mutations (affecting approximately 1,200 genes). Thus, acetylation of histone H4 is interpreted by two mechanisms: a specific mechanism for lysine 16 and a nonspecific, cumulative mechanism for lysines 5, 8, and 12.

Genetic analysis of an Indian family with members affected with juvenile-onset primary open-angle glaucoma

Purpose: Glaucoma is the second leading cause of blindness. In India, ~1.5 million people are blind due to glaucoma. Mutations in the MYOC gene located at the GLC1A locus on chromosome 1q21–q31 have been found in patients with juvenile-onset primary open-angle glaucoma (J-POAG). The purpose of the present study was to identify the genetic cause of glaucoma in a four-generation Indian family affected with J-POAG. Methods: Peripheral blood samples were obtained from individuals for genomic DNA isolation. To determine if this family was linked to the GLC1A locus, haplotyping analysis was carried out using microsatellite markers from the GLC1A candidate region. Exon-specific primers from exon 3 of the MYOC gene were used to amplify DNA samples from individuals. Mutation analysis was carried out using PCR-SSCP and DNA sequence analyses. Results: Pedigree analysis suggested that glaucoma in this family segregated as an autosomal dominant trait. Of six patients, five had J-POAG and one had adult-onset POAG (A-POAG). Haplotype analysis suggested linkage of this family to the GLC1A locus. Mutation and sequence analyses showed a novel missense mutation, c.821C > G (p.P274R), in the C-terminal olfactomedin domain coded by exon 3 of the MYOC gene. One patient was found to be homozygous for this mutation with a severe phenotype. Conclusions: This study reports a novel missense mutation in a four-generation Indian family with all but one member affected with J-POAG. The total number of mutations described so far in the MYOC gene, including the one reported here, is 59 with a clustering of 52 mutations in exon 3.

Mutation analysis of the cathepsin C gene in Indian families with Papillon-Lefèvre syndrome.

BackgroundPLS is a rare autosomal recessive disorder characterized by early onset periodontopathia and palmar plantar keratosis. PLS is caused by mutations in the cathepsin C (CTSC) gene. Dipeptidyl-peptidase I encoded by the CTSC gene removes dipeptides from the amino-terminus of protein substrates and mainly plays an immune and inflammatory role. Several mutations have been reported in this gene in patients from several ethnic groups. We report here mutation analysis of the CTSC gene in three Indian families with PLS.MethodsPeripheral blood samples were obtained from individuals belonging to three Indian families with PLS for genomic DNA isolation. Exon-specific intronic primers were used to amplify DNA samples from individuals. PCR products were subsequently sequenced to detect mutations. PCR-SCCP and ASOH analyses were used to determine if mutations were present in normal control individuals.ResultsAll patients from three families had a classic PLS phenotype, which included palmoplantar keratosis and early-onset severe periodontitis. Sequence analysis of the CTSC gene showed three novel nonsense mutations (viz., p.Q49X, p.Q69X and p.Y304X) in homozygous state in affected individuals from these Indian families.ConclusionsThis study reported three novel nonsense mutations in three Indian families. These novel nonsense mutations are predicted to produce truncated dipeptidyl-peptidase I causing PLS phenotype in these families. A review of the literature along with three novel mutations reported here showed that the total number of mutations in the CTSC gene described to date is 41 with 17 mutations being located in exon 7.

Use of conserved key amino acid positions to morph protein folds.

By using three-dimensional (3D) structure alignments and a previously published method to determine Conserved Key Amino Acid Positions (CKAAPs) we propose a theoretical method to design mutations that can be used to morph the protein folds. The original Paracelsus challenge, met by several groups, called for the engineering of a stable but different structure by modifying less than 50% of the amino acid residues. We have used the sequences from the Protein Data Bank (PDB) identifiers 1ROP, and 2CRO, which were previously used in the Paracelsus challenge by those groups, and suggest mutation to CKAAPs to morph the protein fold. The total number of mutations suggested is less than 40% of the starting sequence theoretically improving the challenge results. From secondary structure prediction experiments of the proposed mutant sequence structures, we observe that each of the suggested mutant protein sequences likely folds to a different, non-native potentially stable target structure. These results are an early indicator that analyses using structure alignments leading to CKAAPs of a given structure are of value in protein engineering experiments.

Pretransplantation pre-S2 and S protein heterogeneity predisposes to hepatitis B virus recurrence after liver transplantation

Liver transplantation (LT) in patients with hepatitis B virus (HBV) infection often is complicated by recurrence of infection despite immunoglobulin treatment. To evaluate whether variability in HBV genomic sequences and the target of antibody to hepatitis B surface antigen action in pre-LT samples may be associated with a high recurrence rate, HBV pre-S/S regions of 14 HBV-positive candidates for LT (in 9 of these patients, HBV infection subsequently recurred) were amplified and sequenced. Two hundred ninety-one mutations in 1,167 sequenced nucleotides (24.9%) were found. Of these, 120 mutations (10.2%) led to an amino-acid change. The only significant difference between patients with and without recurrent disease was in the number of mutations in the pre-S2 region (total mutations, P =.042; missense mutations, P =.012) of pre-LT HBV DNA. In addition, a difference in amino-acid level was present in the pre-S2 region (P =.030). The delay in HBV infection recurrence was proportional to the number of pre-LT HBV mutations in the pre-S2 and S genes: the higher the number, the longer the interval between LT and recurrence of infection (pre-S2, P =.0124; S, P =.0060; total number of mutations in S protein, P =.0421). In conclusion, pre-LT determination of pre-S/S gene sequence variability showed that heterogeneity of the pre-S2 and, to a lesser extent, S genes was associated with a greater chance for HBV recurrence. Modification of B-cell epitopes of S, but especially of pre-S2, protein leading to conformational changes and alterations in the viral encapsidation and secretion process may facilitate HBV recurrence and contribute to the failure of immune globulin therapy.

Sequence Analysis of the Entire Mitochondrial Genome in Parkinson's Disease

The pathogenesis of Parkinson's disease (PD) is largely unknown. Indirect evidence suggests that mutations in mitochondrial DNA (mtDNA) might play a role, but previous studies have not consistently associated any specific mutations with PD. However, these studies have generally been confined to limited areas of the mitochondrial genome. We therefore sequenced the entire mitochondrial genome from substantia nigra of 8 PD and 9 control subjects. Several sequence variants were distributed differently between PD and control subjects, but all were previously reported polymorphisms. Several secondary LHON mutations were found, as well as a number of novel missense mutations, but all were rare and did not differ between PD and control subjects. Finally, PD and control subjects did not differ in the total number of all mutations, nor the total number of missense mutations. Thus, mtDNA involvement in PD, if any, is likely to be complex and should be reconsidered carefully.

Spectrum of mutations in long-QT syndrome genes. KVLQT1, HERG, SCN5A, KCNE1, and KCNE2.

Long-QT Syndrome (LQTS) is a cardiovascular disorder characterized by prolongation of the QT interval on ECG and presence of syncope, seizures, and sudden death. Five genes have been implicated in Romano-Ward syndrome, the autosomal dominant form of LQTS: KVLQT1, HERG, SCN5A, KCNE1, and KCNE2. Mutations in KVLQT1 and KCNE1 also cause the Jervell and Lange-Nielsen syndrome, a form of LQTS associated with deafness, a phenotypic abnormality inherited in an autosomal recessive fashion. We used mutational analyses to screen a pool of 262 unrelated individuals with LQTS for mutations in the 5 defined genes. We identified 134 mutations in addition to the 43 that we previously reported. Eighty of the mutations were novel. The total number of mutations in this population is now 177 (68% of individuals). KVLQT1 (42%) and HERG (45%) accounted for 87% of identified mutations, and SCN5A (8%), KCNE1 (3%), and KCNE2 (2%) accounted for the other 13%. Missense mutations were most common (72%), followed by frameshift mutations (10%), in-frame deletions, and nonsense and splice-site mutations (5% to 7% each). Most mutations resided in intracellular (52%) and transmembrane (30%) domains; 12% were found in pore and 6% in extracellular segments. In most cases (78%), a mutation was found in a single family or an individual.

Conservation of polar residues as hot spots at protein interfaces

A number of studies have addressed the question of which are the critical residues at protein-binding sites. These studies examined either a single or a few protein–protein interfaces. The most extensive study to date has been an analysis of alanine-scanning mutagenesis. However, although the total number of mutations was large, the number of protein interfaces was small, with some of the interfaces closely related. Here we show that although overall binding sites are hydrophobic, they are studded with specific, conserved polar residues at specific locations, possibly serving as energy “hot spots.” Our results confirm and generalize the alanine-scanning data analysis, despite its limited size. Previously Trp, Arg, and Tyr were shown to constitute energetic hot spots. These were rationalized by their polar interactions and by their surrounding rings of hydrophobic residues. However, there was no compelling reason as to why specifically these residues were conserved. Here we show that other polar residues are similarly conserved. These conserved residues have been detected consistently in all interface families that we have examined. Our results are based on an extensive examination of residues which are in contact across protein interfaces. We utilize all clustered interface families with at least five members and with sequence similarity between the members in the range of 20–90%. There are 11 such clustered interface families, comprising a total of 97 crystal structures. Our three-dimensional superpositioning analysis of the occurrences of matched residues in each of the families identifies conserved residues at spatially similar environments. Additionally, in enzyme inhibitors, we observe that residues are more conserved at the interfaces than at other locations. On the other hand, antibody–protein interfaces have similar surface conservation as compared to their corresponding linear sequence alignment, consistent with the suggestion that evolution has optimized protein interfaces for function. Proteins 2000;39:331–342. © 2000 Wiley-Liss, Inc.