ANALYSIS OF INTROGRESSIVE LINES OF INTER-SPECIES PEA HYBRIDS BY BAND COMPOSITION OF SEED PROTEINS

Background. The reproductive incompatibility of cultivated (Pisum sativum) and wild (P. fulvum) pea species determines the difficulties of obtaining hybrids as well as the transfer of valuable wild parent alleles into interspecific hybrids and their use in the breeding process. The aim of the research was a comparative study of protein spectra of pea interspecific hybrids BC2F5 P. sativum x P. fulvum obtained by the authors and their parents. Materials and methods. The band composition of seed proteins in the interspecific hybrids of peas BC2F5, variety Stabil (P. sativum) accession from VIR collection I-609881 (P. fulvum) has been studied. Effectiveness of parent gene transfer determining each polymorphic position of electrophoretic spectrum were evaluated. Results. The ratio of the actual frequencies of the bands of the cultivated and wild parents in the introgression lines corresponded to the expected level in 73% of the positions of the electrophoretic spectrum. The introgression rate of individual seed protein bands from wild parent into interspecific pea hybrids in the absence of selection significantly exceeded the expected level, which may indicate the adaptive value of alleles encoding unique seed protein isoforms. Conclusion. The possibility of introgressive transfer of wild-type alleles to the cultivated genotypes of pea, as well as the presence of identified cultivated isoforms of storage proteins in all studied lines of BC2F5 interspecific hybrids in 88.2% of the polymorphic positions of the electrophoretic spectrum, indicates the possibility of using the wild species P. fulvum in pea breeding.

Polymorphisms Glu23Lys of KCNJ11 gene are associated with Type 2 diabetes in the Kyrgyz Population

Aim — in this study, we investigated whether polymorphisms Glu23Lys in KCNJ11 are associated with Type 2 diabetes mellitus (T2DM) in the Kyrgyz population.Material and methods. We genotyped 287 Kyrgyz individuals. A case—control study was performed, including 178 patients with T2DM (female — 55, male — 123, average age 54±6,6) and 109 non-diabetes controls (female — 48, male — 61, average age 50±8,4). The genotypes of polymorphic position Glu23Lys of KCNJ11 were determined by PCR-RFLP assay.Results. Genotypes Glu23Lys and Lys23Lys, containing the minor allele 23Lys, were more frequent in the group of type 2 diabetes (χ2=4,09; p=0,043). The allele 23Lys of KCNJ11 gene is associated with a high risk of developing type 2 diabetes in the Kyrgyz population [OR=1,46 (1,02—2,07); p=0,036]. Homozygous genotype Glu23Glu and allele Glu23 reduces the risk of developing type 2 diabetes [OR=0,61 (0,37—0,99); p=0,043 and OR=0,69 (0,48—0,98); p=0,037], respectively).Conclusion. Polymorphisms Glu23Lys of KCNJ11 gene are associated with the risk of Type 2 diabetes mellitus in the Kyrgyz Population according to the dominant and additive models of inheritance of the trait. Our results indicate that the allele 23Lys of KCNJ11 gene confers an elevated risk for the development of Type 2 diabetes mellitus in the Kyrgyz population.

High Resolution HLA Typing by Next Generation Exome Sequencing

Abstract 4166 Background High resolution typing of HLA-A, B, C, DQB1 and DRB1 at the allelic level allows transplantation of hematopoietic progenitor cells (HPC) from matched allogeneic donors. The extent of matching has been recognized as one of the most important predictors of survival. High resolution typing relies on Sanger sequence-based typing (SBT) and PCR with sequence-specific primers (SSP), both of which detect nucleotide changes in exons that alter the HLA antigens. Next generation exome sequencing (NGES) offers the potential for rapid and less expensive HLA typing for transplantation of matched allogeneic HPC, but the role of NGES for high resolution HLA typing has not been examined. In this study, we designed a bioinformatic pipeline to account for the hundreds to thousands of alleles at each HLA locus in the population, and used it to explore the possibility of high resolution HLA typing using NGES data. Methods Indexed sequencing libraries were constructed from 3 HapMap samples (NA19129, NA18507, and NA19240), target enriched with SureSelect Human All Exon V3 kit (Agilent Technologies), and sequenced in multiplex as paired-end 2×101 bp reads on an Illumina HiSeq2000. The above process was repeated for NA19240 to assure reproducibility. The NGES-based HLA typing consisted of 3 major steps: 1) Filtering reads: cDNA and genomic sequences of all alleles of each HLA locus [ImMunoGeneTics project (IMGT)/HLA database] were concatenated to form five references, to which input reads were aligned using NovoalignMPI V.2.07.07 with standard options and no more than 1 mismatch per read. 2) Eliminating unsupported alleles: Curated alignment of all alleles of each HLA locus was obtained from IMGT/HLA database. Each polymorphic position was termed a D position. K-mers (a DNA sequence with length k), each containing a D position, was searched against the reads from step 1. Alleles were credited or penalized based on presence or absence of their k-mers in the reads. Alleles with negative overall scores were eliminated and this step was repeated until no elimination occurred. 3) Ranking allele pairs: all possible pairs of candidate alleles from step 2 were evaluated; a pair was credited at each D position if k-mers of both alleles were supported by the reads or penalized if otherwise. The allele pair (or pairs if a tie) ranked at the top was the presumed type of the locus examined. The NGES-based high resolution typing was run on a computing node (IBM x3650-m2). The results were compared with the results of conventional typing (SBT and SSP), followed by examination of the disagreement between the methods and adjustment of k. Results The conventional high resolution typing results comprised 15 pairs of alleles that were uniquely different (one pair of alleles at each locus of HLA-A, B, C, DQB1 and DRB1 for 3 samples). The method was established using the information on HLA-A alleles of NA19129, leaving 14 pairs of alleles for validation. Initial NGES-based typing indicated that the specificity of the method was dependent on k-mer length, with increased length required to eliminate interference from mimicking reads (e.g. mimics of an HLA-C allele). Thus, with a k-mer length of 50 bp, NGES-based typing correctly determined 12 of 14 pairs of alleles, while with k-mer lengths of 60 or 65 bp, it correctly determined 14 of 14 pairs of alleles. All 5 pairs of alleles of NA19240 were also correctly determined with the longer k-mers using reads from the replicate sequencing. After read filtering, the typing took approximately 5 minutes per sample. Conclusion In this first proof-of-concept study, high resolution HLA typing was successfully performed using NGES data. With k-mer lengths of 60 or 65 bp, NGES-based typing correctly identified 100% of tested allele pairs, and demonstrated a similar level of resolution and accuracy as conventional high resolution typing. Note: C.L. and X.Y. contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

Molecular characterization of the honeybee Apis mellifera carnica in Serbia

The sequences COI-COII of the mitochondrial DNA region in honeybee from four geographically distant regions in Serbia (Vrsac, Knjazevac, Kraljevo, and Vranje) are analyzed. The research was conducted on eight different, previously selected honeybee lines preserved (linear selection) in the four reprocenters for queen bees. All four studied honeybee lines differ in morphological and productive traits, each being specific for the corresponding region. In addition to analysis of the mtDNA sequences in Serbian honeybee, a comparative analysis of the phylogenetic group of so far known C2 haplotypes was also performed. The results revealed two novel polymorphic positions in the COI-COII mtDNA region, viz., h2 at position 3474 and l2 at position 3534 (a T nucleotide deletion in both cases) in honeybees from the regions of Vranje and Knjazevac, respectively. Two novel mtDNA haplotypes in the honeybee C2 phylogenetic group, together with C2I (the new polymorphic position l2 and G-A transition at position 3587) and C2J (the new polymorphic position h2), are described. Also, comparative analysis performed on sequences from GenBank data showed a high degree of similarity (similarity index = 99.4%) between the novel C2I mtDNA haplotype and an A. m. cypria haplotype originating from Turkey. Certain domestic Kranjska honeybee populations from Serbia represent an autochthonous gene pool that can be of great importance for further presentation of honeybee biodiversity. The present paper contributes to characterization of mtDNA in honeybee of Serbia.

Substrate Preferences for Antiplasmin Cleaving Enzyme Hydrolysis of Peptides Derived from the α2-Antiplasmin Sequence.

Antiplasmin cleaving enzyme (APCE) is a proteinase that specifically, but slowly cleaves the Pro-Asn bond in long-form α2-antiplasmin (Met-α2AP) in human plasma. This slow cleavage produces a steady-state plasma mixture of Met-α2AP and an N-terminally shortened form of antiplasmin, Asn-α2AP. The Asn-α2AP form crosslinks to fibrin ~13-fold faster than Met-α2AP. A faster crosslink rate causes a greater number of antiplasmin molecules to become bound during fibrin formation, thereby enhancing resistance to fibrinolysis. Inhibition of plasma APCE may decrease the number of antiplasmin molecules crosslinked and result in clots that are more easily removed during fibrinolysis. Therefore, an inhibitor specific for APCE could potentially be used to regulate fibrinolysis. Human Met-α2AP exists in two polymorphic forms at position six in the mature sequence, with arginine predominant, and tryptophan accounting for a lesser percentage. We have determined the relative cleavage rates of synthetic peptides from a peptide library that span the cleavage site. The peptides contained all common amino acids except cysteine in the polymorphic position (P7 position). Arg was the optimal amino acid in this position with a relative cleavage rate ~5–10-fold faster than other amino acids except Lys, which was ~70% of the Arg rate. The P7 position Arg enhancement was also observed when Arg was in the P6 or P5 position, but no enhancement was observed when Arg was moved to positions P8, P4, P3 or P2. It was also determined that APCE is preferentially an endoproteinase rather than an aminodipeptidase, with a 10-fold greater rate of hydrolysis of the internal Pro-Asn bond in the Met-α2AP 1–17 peptide sequence MEPLGRQLTSGP-NQEQV over the Pro-Asn bond penultimate to the amino-terminal bond in the Met-α2AP 11–27 peptide sequence GP-NQEQVSPLTLLKLGN in peptide hydrolysis experiments. We conclude that APCE inhibitors designed with a positive charge placed upstream of the Pro-X scissile bond equivalent to five to seven amino acid residues may prove to be highly potent and specific. In addition, such inhibitors should also prove useful for blocking the activity of the closely related enzyme fibroblast activation protein. This work was supported by NIH grant HL072995.

A Polymorphic Oncofetal Antigen Recognized by CD8+ CTL from Two Patients Experiencing Regression of Metastatic Renal Cell Carcinoma after Allogeneic HCT.

Regression of metatstatic renal cell carcinoma (RCC) has been observed in 8–57% of patients undergoing nonmyeloablative allogeneic hematopoietic cell transplant (HCT) from HLA-matched donors. Tumor regression in this setting typically occurs after complete donor T cell engraftment is established, and is correlated with the development of GVHD. We previously isolated from two patients experiencing regression of metastatic RCC after nonmyeloablative allogeneic HCT CD8+ CTL clones that recognized a RCC-associated minor histocompatibility (H) antigen presented by HLA-A*0201, and localized the gene encoding this antigen to chromosome 19q. To identify the gene that encodes this antigen, a cDNA expression library made from a HLA-A*0201+ minor H antigen-positive tumor cell line was screened using a transfection assay in COS-7 cells. After two rounds of screening, a 1.4 kb cDNA was identified that stimulated HLA-A*0201-dependent cytokine release from minor H antigen-specific CTL. Sequencing showed that this cDNA was derived from the MGC13170 gene on chromosome 19q, within the region of significant linkage. Sequencing of MGC13170 alleles in minor H antigen-positive and minor H antigen-negative cells revealed six single nucleotide polymorphisms (SNPs), inherited as a haplotype and all previously identified, that determined susceptibility or resistance to lysis by minor H antigen-specific CTL. Analysis of 5′ and 3′ truncation constructs derived from the MGC13170 cDNA identified an interval encoding a 48-residue open reading frame that contained the epitope and spanned one of the six SNPs (dbSNP rs3745526) that had been shown to correlate with CTL recognition. Further analysis of minigenes spanning this nonsynonymous T↔A SNP identified an interval containing T at the polymorphic position and encoding a predicted 11-residue peptide that stimulated maximal HLA-A2-dependent cytokine release from minor H antigen-specific CTL. The T↔A SNP is predicted to create a Ser↔Thr polymorphism in the encoded peptide. A synthetic 11-mer peptide containing Ser at the polymorphic residue, but not the homologous peptide containing Thr at this position, was recognized by minor H antigen-specific CTL when pulsed onto minor H antigen-negative HLA-A*0201+ cells, thus demonstrating that the T allele of MGC13170 encodes the minor H antigenic peptide. The function of MGC13170 is unknown, but a previous study (Zhou et al., Ai Zheng21:341–345, 2002) suggested a role in resistance of tumor cells to chemotherapeutic agents. The range of tissues in which the epitope-encoding MGC13170 sequence is expressed was evaluated in silico by probing the translated human EST database with the predicted sequence of the 48-residue polypeptide encoded by the epitope-containing ORF. Of the several hundred ESTs identified that encoded the Ser- or Thr-containing peptide sequence, 72% were derived from tumor cells or cell lines (including RCC), 5% from embryonic or fetal tissues, 9% from embryonic stem cells, and 11% from normal tissues. Thus, MGC13170 encodes a novel HLA-A*0201-restricted minor H antigen that is expressed in a wide variety of neoplastic, embryonic, and fetal cells. Further studies of MGC13170 as a possible target for antitumor immune responses after allogeneic HCT are underway.

Paraoxonase-1 promoter haplotypes and serum paraoxonase: a predominant role for polymorphic position −107, implicating the Sp1 transcription factor

Accumulating data suggest that paraoxonase-1 (PON1) is a primary determinant of the antioxidant and anti-inflammatory capacities of high-density lipoproteins (HDLs). Variations in HDLs and PON1 have been shown to influence the functions of both. There is a wide spectrum of serum PON1 mass in humans, to which promoter polymorphisms make an important contribution. The present studies attempted to define: (i) the relevance in vivo of promoter polymorphisms by analysing haplotype structure; and (ii) molecular mechanisms implicated in promoter activity. Highly significant differences (P<0.0001) in serum mass and activity were observed as a function of haplotype sequence. Of three promoter polymorphisms (−107, −824 and −907), the −107 site was shown to be of predominant importance to serum PON1. Significant increases in serum PON1 mass and activities between haplotype subgroups could be explained by unit increases in the number of high-expresser variants of the −107 site (−107C) alone. No significant contribution was observed for the −824 and −907 sites. The coding-region Leu55→Met (L55M) polymorphism made an independent contribution to serum PON1 mass, which may account for variations in serum PON1 mass and activity within haplotype subgroups defined by the −107 site. A molecular basis for the effect of the −107 polymorphism on serum PON1 was indicated by the greater affinity of the high-expresser variant (−107C) for hepatocyte nuclear extracts, indicating higher affinity for transcription factors. Competition studies with oligonucleotides representing the consensus (and mutated) sequence for Sp1, and the use of Sp1 antibodies, confirmed formation of complexes between the transcription factor and the PON1 promoter during incubation with nuclear extracts. The data underline the importance of the region containing the C(−107)T polymorphism for gene expression in vivo. Differences in the affinity of the −107C and −107T polymorphic fragments for nuclear extracts have been demonstrated, and coincide with their impact on gene expression. A potential role for the transcription factor Sp1 has been demonstrated, which is consistent with the disruption of an Sp1 recognition sequence by the −107 polymorphism.

Characterization of genetic polymorphism in the goat β-lactoglobulin gene

Two new variants have been detected and characterized for the goat β-lactoglobulin gene at the cDNA level and confirmed at the genomic level. The two polymorphisms are located on exon 7 of the gene. One of the polymorphic sites is produced by a single nucleotide substitution in position +4601, allowing a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) genotyping procedure to be developed using SacII restriction enzyme. The other polymorphic position contains a 10 bp long insertion at position +4641 that can be detected by capillary electrophoresis of the PCR product amplified with a fluorescent primer. The association of these two polymorphisms was also investigated, resulting in the description of two new alleles. Both of these contained the point mutation at the SacII site, with or without the 10 bp insertion at position +4641. The distribution of these new polymorphisms was studied in a population of males of four different goat breeds. The gene frequencies for these variants were similar in Spanish and French breeds.