Produce Amino Acid Substitutions Research Articles

A genomic variant of ALPK2 is associated with increased liver fibrosis risk in HIV/HCV coinfected women.

HIV coinfection is associated with more rapid liver fibrosis progression in hepatitis C (HCV) infection. Recently, much work has been done to improve outcomes of liver disease and to identify targets for pharmacological intervention in coinfected patients. In this study, we analyzed clinical data of 1,858 participants from the Women's Interagency HIV Study (WIHS) to characterize risk factors associated with changes in the APRI and FIB-4 surrogate measurements for advanced fibrosis. We assessed 887 non-synonymous single nucleotide variants (nsSNV) in a subset of 661 coinfected participants for genetic associations with changes in liver fibrosis risk. The variants utilized produced amino acid substitutions that either altered an N-linked glycosylation (NxS/T) sequon or mapped to a gene related to glycosylation processes. Seven variants were associated with an increased likelihood of liver fibrosis. The most common variant, ALPK2 rs3809973, was associated with liver fibrosis in HIV/HCV coinfected patients; individuals homozygous for the rare C allele displayed elevated APRI (0.61, 95% CI, 0.334 to 0.875) and FIB-4 (0.74, 95% CI, 0.336 to 1.144) relative to those coinfected women without the variant. Although warranting replication, ALPK2 rs3809973 may show utility to detect individuals at increased risk for liver disease progression.

Whole-Exome Sequencing in Evaluation of Thrombophilia: Characterization of Novel Genetic Variants

Abstract Venous thromboembolism (VTE), defined as deep venous thrombosis and pulmonary embolism, is a cause of significant morbidity and mortality worldwide, with an overall incidence of about 10,000,000 cases per year. The majority of VTE is attributable to genetic factors, yet the five major heritable thrombophilias of factor V Leiden, prothrombin gene mutation, antithrombin deficiency, protein C deficiency, and protein S deficiency comprise only a minority of VTE cases, suggesting that further genetic factors must contribute to VTE risk. We performed whole-exome sequencing (WES) in 106 patients with VTE, focusing our analysis on a 55-gene extended thrombophilia panel comprising genes previously reported in next-generation sequencing studies to be associated with VTE. In our cohort, we identified a probable disease-causing genetic variant or variant of unknown significance (VUS) in approximately 60% of study patients. We then selected several novel VUS involving genes with known roles in hemostasis for further characterization to determine their biological and pathological significance in thrombophilia. Multiple sequence alignment analysis showed that several nonsynonymous variants produced amino acid substitutions in highly conserved regions of proteins relevant to hemostasis and thrombosis, including thrombomodulin (TM), heparin cofactor II, ADAMTS13, and von Willebrand factor (vWF). To evaluate the theoretical effects of selected VUS on thrombosis susceptibility, we visualized proteins using PyMol and performed in silico mutagenesis modeling to evaluate for structural alterations that can predict functional and physiological consequences. Our analyses showed that several variants caused conformational changes predicted to either result in a deleterious effect on enzymatic activity or prevent substrate binding to the active site. For example, variants in TM (P401L and V454A) are predicted to decrease thrombin binding and protein C activation, whereas a C668R variant in the spacer domain of ADAMTS13 is predicted to prevent binding to vWF, resulting in ultra-large vWF multimers. We plan to use these results in combination with clinical laboratory-based coagulation testing and family studies to determine pathogenicity of specific variants in thrombosis and thrombophilia. Overall, these findings indicate that WES is a potentially useful tool for inherited thrombophilia and can provide insight into the genetic factors contributing to VTE.

Genetic variability and phylogeny analysis of partial L1 gene of human papillomavirus variants in Buenos Aires, Argentina.

In the present study, the molecular characterization of HPV variants 16, 18, 31, 58, 6 and 11 within the MY06/MY11 L1 genomic region was performed in 128 sequences. For HPV 16, all of the sequences analyzed had a 3 nucleotide insertion resulting in the insertion of serine in the L1 protein sequence; and 4 sequences had at least one single nucleotide polymorphism (SNP). Twelve base substitutions were detected in HPV 58, 6 SNPs produced amino acid changes, and the other SNPs detected were found to be silent mutations. For HPV 31, 25 SNPs were detected as silent mutations. Of the 8 SNPs detected on HPV 18, three produced amino acid changes, the remaining SNPs detected were silent mutations. For HPV 6, 10 SNPs were detected and none of them produced amino acid changes. From the 16 sequences analyzed for HPV 11, two SNPs were detected and neither of them produced amino acid substitutions. Phylogenetic trees were constructed for HPV 16, HPV 18, HPV 31, HPV 58, HPV 6 and HPV 11. In the current study 8 new variants were identified based on sequencing of the L1 region. Changes in the L1 region of the HPV genome may be important for discriminating the infectious potential of different variants, as well as in defining epitopes relevant to vaccine design. The findings of this study indicate that there are new variants of HPV circulating in Argentina, which need to be confirmed by further analyses of the complete HPV genome.

Ribosomal cold-adaptation: Characterization of the genes encoding the acidic ribosomal P0 and P2 proteins from the Antarctic ciliate Euplotes focardii

Molecular adaptation at low temperature requires specificities represented mainly by modifications in the gene sequence and consequently in the protein primary structure. To characterize the molecular mechanisms responsible for ribosome cold-adaptation, we compared the ribosomal P0 and P2 genes from the Antarctic ciliate Euplotes focardii with homologous genes from mesophilic organisms, including the ciliates Tetrahymena thermophila and non cold-adapted Euplotes species. This analysis revealed the presence of non synonymous mutations unique to E. focardii. In the P0 protein the mutations produced amino acid substitutions that increased the molecular flexibility that may facilitate a conformational adjustment associated with the interaction with the GTPase center of the large subunit rRNA, and increased the hydrophobicity of the region involved in the interaction with P1/P2 heterodimer, probably to keep associated the ribosomal stalk in the cold. In the P2 protein the mutations produced amino acid substitutions that increased the N-terminus flexibility, which may facilitate interactions with P1 protein in the formation of the heterodimer, and reduced the mobility of the C-terminus, to stabilize the stalk during ribosomal activity. Finally, P proteins appeared to be valid markers for investigating the phylogenetic origin of early eukaryotes.

Identification and characterization of single-nucleotide polymorphisms in MCH-R1 and MCH-R2.

To identify and functionally characterize single-nucleotide polymorphisms (SNPs) in melanin-concentrating hormone (MCH)-R1 and -R2. The entire coding regions and intron/exon splice junction regions of MCH-R1 and MCH-R2 were sequenced from anonymous white (n=45) and African-American (n=46) individuals. DNA was analyzed, and SNPs were identified using Phred, Phrap, and Consed software. DNA constructs containing MCH-R1 and MCH-R2 SNPs were generated and expressed in CHO cells. The effect of the SNPs in MCH-R1 and MCH-R2 were assessed in receptor binding assays and functional assays measuring changes in intracellular cAMP and Ca2+ levels. We identified 12 SNPs in the MCH-R1 gene. Two of these SNPs are in coding regions, and one produces an arginine-for-glycine substitution at residue 34 in the MCH-R1 sequence. This SNP is present at a minor allele frequency of 15% in the African-American population tested in this study. We identified eight SNPs in the MCH-R2 gene. Four of these SNPs are in coding regions, and two produce amino acid substitutions. Lysine substitutes for arginine at residue 63 of the African-American population, and glutamine substitutes for arginine at residue 152 in whites (minor allele frequency of 2% for both SNPs). No changes in receptor binding or functional signaling were observed with the SNP mutations in MCH-R1 or MCH-R2. These data indicate that potential therapeutics designed to act at the MCH receptor are unlikely to have altered effects in subpopulations that express variant forms of MCH-R1 or MCH-R2.

A paradigm for single nucleotide polymorphism analysis: the case of the acetylcholinesterase gene.

Acetylcholinesterase (AChE) plays a crucial physiological role in termination of impulse transmission at cholinergic synapses through rapid hydrolysis of acetylcholine. It is a highly conserved molecule, and only a few naturally occurring genetic polymorphisms have been reported in the human gene. The goal of the present study was to make a systematic effort to identify natural single nucleotide polymorphisms (SNPs) in the human ACHE gene. To this end, the genomic coding sequences for acetylcholinesterase of 96 unrelated control individuals from three distinct ethnic groups were analyzed. A total of 13 ACHE SNPs were identified, 10 of which are newly described, and five that should produce amino acid substitutions [c.101G>A (p.Arg34Gln), c.169G>A (p.Gly57Arg), c.1031A>G (p.Glu344Gly), c.1057C>A (p.His353Asn), and c.1775C>G (p.Pro592Arg)]. Population frequencies of 11 of the 13 SNPs were established in four different populations: African Americans, Ashkenazi Jews, Sephardic Jews, and Israeli Arabs; 15 haplotypes and five ethnospecific alleles were identified. The low number of SNPs identified until now in the ACHE gene is ascribed to technical hurdles arising from the high GC content and the presence of numerous repeat sequences, and does not reflect its intrinsic heterozygosity. Among the SNPs resulting in an amino acid substitution, three are within the mature protein, mapping on its external surface: they are thus unlikely to affect its catalytic properties, yet could have antigenic consequences or affect putative protein-protein interactions. Furthermore, the newly identified SNPs open the door to a study of the possible association of AChE with deleterious phenotypes-such as adverse drug responses to AChE inhibitors employed in treatment of Alzheimer patients and hypersensitivity to pesticides.

Analysis of the p63 gene in classical EEC syndrome, related syndromes, and non-syndromic orofacial clefts

EEC syndrome is an autosomal dominant disorder with the cardinal signs of ectrodactyly, ectodermal dysplasia, and orofacial clefts. EEC syndrome has been linked to chromosome 3q27 and heterozygous p63 mutations...

Genetic polymorphism of the caprine kappa casein gene.

Polymorphism in the goat kappa casein gene was studied using the base excision sequence scanning (BESS) method and sequencing. Seven polymorphic sites, corresponding to single nucleotide transversions were detected. Three of these were silent mutations while the other four produced amino acid substitutions. The association between these polymorphic sites was investigated, which resulted in the identification of three goat kappa casein alleles, designated A, B, and C. Protocols for rapid genotyping of the C variant were developed by polymerase chain reaction-restriction fragment length polymorphism using Alw44I and BseNI restriction endonucleases. The occurrence of this allele was found to be very low in Spanish breeds but more frequent in the French Saanen goat. Further studies among different goat populations are necessary to establish the distribution of these alleles and their effects on the quality and functional properties of milk.

Mutated interleukin-5 monomers are biologically inactive

Interleukin-5 contains only two cysteine residues both of which appear to be involved in the dimerisation of the molecule to form a disulphide-linked homodimer (Minamitake et al., J. Biochem. 107, 292–297, 1990). However, it remains unclear whether this linkage is necessary for the bioactivity of this cytokine. Site-directed mutagenesis was used to produce amino acid substitutions of either or both of the cysteines. The mutant proteins were all biologically inactive monomers, however when the two single mutant constructs were co-transfected, biologically active IL5 was produced. This is consistent with the dimer forming in a head-to-tail configuration.

The evolution of muscular parvalbumins investigated by the maximum parsimony method.

Phylogenetic trees requiring the lowest sum of nucleotide replacements and gene duplicative events were constructed from the amino acid sequence data on ten gnathostome parvalbumins (PAR) and two related myofibrillar proteins troponin-C (TNC) and myosin alkali-light-chain (ALC). The origin and differentiation of the structural domains within these proteins were also investigated by the maximum parsimony method and by an alignment statistic for identifying evolutionarily related protein sequences. The results suggest, in agreement with the Weeds-McLachlan model, that tandem duplications in a precursor gene caused a primordial one-domain polypeptide (consisting of two helices with a calcium binding region in between) to double and then quadruple in size. Duplications of the gene coding for this four domain (I–II–III–IV) protein in an early metazoan, pre-gnathostome lineage gave rise to the separate loci for TNC, ALC, and PAR. TNC, which alone retained the Ca-binding function in each of its four domains, evolved much more slowly than either the ALC or PAR lineages. In the PAR lineage the I–II–III–IV structure was degraded, presumably by a partial gene deletion, to the II–III–IV structure during descent to the gnathostome ancestor of parvalbumins. Also during this period the mid region in domain II lost its Ca-binding function and, as it did so, evolved at an accelerated rate over other regions, a pattern indicative of positive selection for a change in function. In turn, from the gnathostome ancestor to the present, the mid regions of domains III and IV, which each retained Ca-bindung function, evolved much more slowly than other regions, a pattern indicative of stabilizing selection for preservation of function. Between the gnathostome and teleost-tetrapod ancestor a gene duplication separated the parvalbumins into anα-lineage and aβ-lineage. During this early vertebrate period PAR genes evolved at the extremely fast rate of 89 nucleotide replacements per 100 codons per 108 years (i.e. 89 NR %), but from the teleost-tetrapod ancestor to the present, bothα- andβ-PAR lineages evolved at a much slower rate, about 8 NR %. The use ofβ-parvalbumins as phylogenetic markers was complicated by presumptive evidence that paralogous (i.e. duplication dependent) gene lineages occur within this group. As a final point, in the genealogy of TNC, ALC, and PAR lineages, a non-random pattern of nucleotide replacements was observed between the reconstructed ancestral and descendant mRNA sequences. The pattern was similar to that observed for other protein genealogies and seems to reflect a bias in the genetic code for guanine to adenine and adenine to guanine transitions (especially at the first nucleotide position of the RNA codons) to produce amino acid substitutions which are compatible with the preservation of protein three-dimensional structure.