Prebiotic N-(2-Aminoethyl)-Glycine (AEG)-Assisted Synthesis of Proto-RNA?

Quantum mechanical calculations are used to explore the thermodynamics of possible prebiotic synthesis of the building blocks of nucleic acids. Different combinations of D-ribofuranose (Ribf) and N-(2-aminoethyl)-glycine (AEG) (trifunctional connectors (TCs)); the nature of the Ribf, its anomeric form, and its ring puckering (conformation); and the nature of the nucleobases (recognition units (RUs)) are considered. The combinatorial explosion of possible nucleosides has been drastically reduced on physicochemical grounds followed by a detailed thermodynamic evaluation of alternative synthetic pathways. The synthesis of nucleosides containing N-(2-aminoethyl)-glycine (AEG) is predicted to be thermodynamically favored suggesting a possible role of AEG as a component of an ancestral proto-RNA that may have preceded today's nucleic acids. A new pathway for the building of free nucleotides (exemplified by 5'-uridine monophosphate (UMP)) and of AEG dipeptides is proposed. This new pathway leads to a spontaneous formation of free UMP assisted by an AEG nucleoside in an aqueous environment. This appears to be a workaround to the "water problem" that prohibits the synthesis of nucleotides in water.

Next-generation sequencing can reveal in vitro-generated PCR crossover products: some artifactual sequences correspond to HLA alleles in the IMGT/HLA database.

The high-resolution human leukocyte antigen (HLA) genotyping assay that we developed using 454 sequencing and Conexio software uses generic polymerase chain reaction (PCR) primers for DRB exon 2. Occasionally, we observed low abundance DRB amplicon sequences that resulted from in vitro PCR 'crossing over' between DRB1 and DRB3/4/5. These hybrid sequences, revealed by the clonal sequencing property of the 454 system, were generally observed at a read depth of 5%-10% of the true alleles. They usually contained at least one mismatch with the IMGT/HLA database, and consequently, were easily recognizable and did not cause a problem for HLA genotyping. Sometimes, however, these artifactual sequences matched a rare allele and the automatic genotype assignment was incorrect. These observations raised two issues: (1) could PCR conditions be modified to reduce such artifacts? and (2) could some of the rare alleles listed in the IMGT/HLA database be artifacts rather than true alleles? Because PCR crossing over occurs during late cycles of PCR, we compared DRB genotypes resulting from 28 and (our standard) 35 cycles of PCR. For all 21 cell line DNAs amplified for 35 cycles, crossover products were detected. In 33% of the cases, these hybrid sequences corresponded to named alleles. With amplification for only 28 cycles, these artifactual sequences were not detectable. To investigate whether some rare alleles in the IMGT/HLA database might be due to PCR artifacts, we analyzed four samples obtained from the investigators who submitted the sequences. In three cases, the sequences were generated from true alleles. In one case, our 454 sequencing revealed an error in the previously submitted sequence.

Increased HLA class I and II diversity as 72 novel alleles are identified in volunteers for the National Marrow Donor Program Registry in 2010.

Seventy-two novel human leukocyte antigen (HLA) class I and class II alleles are described from volunteers for the 'Be The Match Registry®': 17 HLA-A alleles, 12 HLA-C alleles, 31 HLA-B alleles and 12 HLA-DRB1 alleles. Forty-six (≈ 64%) of the 72 novel alleles are single-nucleotide substitution variants when compared with their most homologous allele. Five of these single-nucleotide variants are silent substitutions and one creates a non-expressed allele (B*44:108N). The remaining novel alleles differ from their most similar allele by two to five nucleotide substitutions. One of the novel HLA-C alleles (C*07:150Q) is of questionable expression due to an insertion of 21 nucleotides starting at codon 143 that adds seven amino acids to exon 3. An inter-locus gene conversion may have created the novel allele HLA-A*23:31 that shares its codon differences with HLA-B*07:28. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-A (codons 116 and 150), HLA-C (codon 114), HLA-B (codons 11, 21, 35, 42, 48, 73, 98 and 170) and HLA-DRB1 (codon 29) loci.

On the prebiotic selection of nucleotide anomers: A computational study.

Present-day known predominance of the ß- over the α-anomers in nucleosides and nucleotides emerges from a thermodynamic analysis of their assembly from their components, i.e. bases, sugars, and a phosphate group. Furthermore, the incorporation of uracil into RNA and thymine into DNA rather than the other way around is also predicted from the calculations. An interplay of kinetics and thermodynamics must have driven evolutionary selection of life's building blocks. In this work, based on quantum chemical calculations, we focus on the latter control as a tool for "natural selection".

Ninety-six novel HLA class I and II alleles identified in volunteers for the National Marrow Donor Program Registry in 2009.

Ninety-six novel human leukocyte antigen (HLA) class I and class II alleles are described from volunteers for the 'Be The Match Registry®': 15 HLA-A alleles, 11 HLA-C alleles, 36 HLA-B alleles and 34 HLA-DRB1 alleles. Sixty-eight (∼71%) of the 96 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Twenty-three of these single nucleotide variants are silent substitutions and one creates a non-expressed allele (B(*) 27:59N). The remaining novel alleles differ from their most similar allele by two to five nucleotide substitutions. One of the novel HLA-A alleles (A(*) 11:52Q) is of questionable expression because of a deletion of codon 11. Three of the novel alleles encode amino acid changes at codons 63 (HLA-C), 88 (HLA-B) and 79 (HLA-DRB1) which have not previously been reported to be polymorphic in these loci. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-A (codons 115, 149 and 168), HLA-C (codon 90), HLA-B (codons 23, 44, 70, 120 and 153) and HLA-DRB1 (codon 88) loci.

Seventy-eight novel HLA class I and II alleles identified during routine registry typing in 2008 and 2009.

Seventy-eight novel human leukocyte antigen (HLA) class I and class II alleles are described: 19 HLA-A alleles, 30 HLA-C alleles, 21 HLA-B alleles, 7 HLA-DRB1 alleles and 1 HLA-DPB1 allele. Eight (∼10%) of the novel alleles were found in more than one individual and may be more common in the population. Sixty-two (∼80%) of the 78 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Twelve of these single nucleotide variants are silent substitutions and one creates a null allele (C*08:26N). One of the novel HLA-C alleles, C*03:58, is a hybrid that likely resulted from an intra-locus recombination. The remaining novel alleles differ from their most similar allele by two to seven nucleotide substitutions. Four of the novel HLA-C alleles encode amino acid changes at codons 41, 42, 55 or 200 which have not previously been reported to be polymorphic. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-A (codons 55 and 120), HLA-C (codons 151, 153 and 176), HLA-B (codons 31 and 84) and HLA-DRB1 (codon 47) loci.

Novel HLA class I and II alleles identified during routine registry typing in 2010.

Twenty-one novel human leukocyte antigen (HLA) class I and class II alleles are described: seven HLA-A alleles, five HLA-C alleles, five HLA-B alleles and four HLA-DRB1 alleles. Seventeen (∼81%) of the 21 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Nine of these single nucleotide variants cause an amino acid substitution, while eight are silent substitutions. The remaining novel alleles differ from their most similar allele by two to three nucleotide substitutions. One novel HLA-C locus allele encodes an amino acid change at codon 10 that previously has not been reported to be polymorphic for this locus. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-A (codons 24 and 156) and HLA-B (codons 40 and 115) loci.

Sixty-five novel alleles at the HLA-A, -B, and -DRB1 loci identified from National Marrow Donor Program volunteer donors.

Sixty-five novel human leukocyte antigen (HLA) alleles are described from volunteer donors of the 'Be The Match Registry': 29 HLA-A alleles, 24 HLA-B alleles, and 12 HLA-DRB1 alleles. Eight (∼12%) of the novel alleles were found in more than one individual and may be more common in the population. Forty (∼62%) of the 65 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Two of these single nucleotide variants are silent substitutions and one creates a null allele. The remaining novel alleles differ from their most similar allele by 2-10 nucleotide substitutions. Some of the novel alleles encode amino acid changes at codons not previously reported to be polymorphic, such as codons 23, 93, 129, and 155 in HLA-A alleles and codon 3 in HLA-B alleles.

Characterization of new HLA-B and -DRB1 alleles from Singapore.

Six novel alleles, three human leukocyte antigen (HLA)-B and three HLA-DRB1 alleles, are described. Five of the variants are single nucleotide substitutions from their most homologous allele, of which three result in amino acid changes (B*3572, *9509 and DRB1*1157) and two are silent substitutions (B*370103 and DRB1*150204). DRB1*0462 differs by three nucleotide substitutions that alter two amino acids.

Characterization of 104 novel alleles at the HLA-A, -B, and -DRB1 loci from National Marrow Donor Program volunteer donors.

One hundred and four novel human leukocyte antigen (HLA) alleles are described from volunteer donors of the National Marrow Donor Program: 37 HLA-A alleles, 37 HLA-B alleles, and 30 HLA-DRB1 alleles. Seventeen ( approximately 16%) of the novel alleles were found in multiple individuals and likely are relatively common in the population. Seventy-two ( approximately 69%) of the 104 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Nine of these single nucleotide variants are silent substitutions and three create null alleles. The remaining novel alleles differ from their most similar allele by two to seven nucleotide substitutions. Some of the novel alleles encode amino acid changes at positions not previously reported to be polymorphic, such as codons 6 and 11 in HLA-A alleles and codons 5, 105, and 141 in HLA-B alleles. Interestingly, one of the novel HLA-DRB1 alleles (*1471) has a change that is not the typical glycine/valine dimorphism at codon 86, which plays a key role in peptide binding to DR molecules. This is only the second DRB1 allele described that encodes an amino acid other than glycine or valine at this position.

Evaluating the potential impact of mismatches outside the antigen recognition site in unrelated hematopoietic stem cell transplantation: HLA-DRB1*1454 and DRB1*140101.

DNA sequencing of 268 individuals drawn from four US populations carrying two unresolved DRB1*14 alleles differing only outside the antigen recognition site identified DRB1*1454 in the majority. A database of 4222 human leukocyte antigen (HLA)-matched hematopoietic stem cell transplantation donor-recipient pairs was queried to determine the number likely mismatched for DRB1*140101/DRB1*1454 but matched for class I loci. A power calculation suggests that more than 88,000 transplants among European Americans will be needed to identify sufficient 7/8 allele-matched pairs to evaluate the impact of the DRB1*140101/DRB1*1454 mismatch on transplant outcome. Molecular modeling of the HLA-DR interaction with the T-cell receptor and with CD4 suggests that the amino acid substitution distinguishing the two alleles will have minimal impact on allorecognition.

Description of novel class I alleles encountered during routine registry typing in 2007.

Twenty-six novel human leukocyte antigen (HLA) class I alleles are described: 3 HLA-A alleles, 19 HLA-B alleles and 4 HLA-C alleles. Only one of the novel alleles (HLA-B*0753) was found in multiple individuals and likely is not uncommon in the population. Nineteen (approximately 70%) of the 26 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Four of these single nucleotide variants are silent substitutions, and one creates a null allele. The remaining novel alleles differ from their most similar allele by two to six nucleotide substitutions. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-B lows (codons 30, 67 and 72), while HLA-Cw*0347 encodes an amino acid change at a position not previously reported to be polymorphic for this locus.

Desmoglein-1-specific T lymphocytes from patients with endemic pemphigus foliaceus (fogo selvagem).

Fogo selvagem (FS), the endemic form of pemphigus foliaceus, is a cutaneous autoimmune disease characterized by subcorneal blistering of the epidermis and the production of autoantibodies against the desmosomal antigen desmoglein-1 (Dsg1). Previously, we showed that mice injected with autoantibodies from FS patients develop a skin disease that reproduces the clinical, histological, and immunological features of FS, indicating that autoantibodies play an essential role in the development of this disease. The purpose of this study was to characterize the autoimmune T-cell response associated with FS. We provide here the first evidence, to our knowledge, that the great majority of FS patients have circulating T lymphocytes that specifically proliferate in response to the extracellular domain of Dsg1. Long-term T cells developed from these patients also responded to Dsg1, and this antigen-specific response was shown to be restricted to HLA-DR molecules. These Dsg1-reactive FS T cells exhibited a CD4-positive memory T-cell phenotype and produced a T helper 2-like cytokine profile. These findings represent the initial steps in defining the role of T cells in FS autoimmunity.

Epitopes targeted by bullous pemphigoid T lymphocytes and autoantibodies map to the same sites on the bullous pemphigoid 180 ectodomain.

Bullous pemphigoid is a blistering skin disease characterized by autoantibodies directed against the NC16A domain of bullous pemphigoid 180 (collagen XVII), a transmembrane protein of epidermal basal cells. Passive transfer studies in mice have shown that antibodies that bind to this immunodominant region of bullous pemphigoid 180 are capable of inducing a skin disease that closely mimics bullous pemphigoid, supporting the hypothesis that epitopes within NC16A are involved in the pathogenesis of bullous pemphigoid. In this study, we examined the autoimmune T cell response in bullous pemphigoid patients. T cells from eight of 12 bullous pemphigoid patients, all of whom had circulating anti-bullous pemphigoid 180 autoantibodies, showed a specific proliferative response to recombinant forms of NC16A. T cell lines and clones developed from four of these patients recognize the same NC16A peptides as those targeted by autoantibodies from the corresponding individuals. These NC16A-responding T lymphocytes express alpha/beta T cell receptors and CD4 memory T cell surface markers and exhibited a Th1/Th2 mixed cytokine profile that may support the production of antibodies. This new information will aid in defining the key steps involved in the development of the autoimmune response in bullous pemphigoid.

Associations of MHC class II alleles with insulin-dependent diabetes mellitus (IDDM) in patients from North India.

Thirty-four insulin-dependent diabetes mellitus (IDDM) patients from North India were studied with respect to their HLA class II alleles including those of the DRB1, DQA1, DQB1 and DPB1 loci, using the polymerase chain reaction (PCR) and hybridization with sequence-specific oligonucleotide probes (SSOP). They were compared with the class II alleles of 94 normal adult controls from the same ethnic background. The results show a statistically significant increase of DRB1*03011 (p < 0.00001), DQB1*0201 (p < 0.007), DQA1*0501 (0.0027) and DPB1*2601 (p < 0.0042) in patients compared to controls. DR*04 was not significantly increased. However, homozygosity for DRB1*03011 was increased more than expected. DRB1*1501 and *1502 did not show a significant decrease in the patients. However, DRB1*0701 was decreased significantly, but this difference did not remain significant when the p value was corrected for the number of alleles tested. Similarly, DPB1*2601 was increased significantly in the patients but did not remain significant after p was corrected for the number of alleles tested. However, DPB1*2601 was increased, and remained significant after correction, in patients not having HLA-DR3. We also studied the possible role of aspartic acid at codon 57 of the DQ beta chain in protection against development of diabetes, and arginine at codon 52 of the DQ alpha chain in susceptibility. We observed an increase in non-Asp57 alleles in DQ beta and Arg52 in DQ alpha in the patients, however, this effect seems to be due to the fact that the most prevalent haplotype in diabetic patients: DRB1*03011-DQA1*0501-DQB1*0201, has DQB1 and DQA1 alleles which carry non-Asp57 and Arg52, respectively.

Typing for all known MICA alleles by group-specific PCR and SSOP.

Major histocompatibility complex class I chain-related gene (MICA) is a recently discovered polymorphic gene in the HLA region expressed mainly by certain epithelial cells, keratinocytes, endothelial cells, fibroblasts, and monocytes. MICA is structurally quite different from the HLA class I genes and is potentially associated with some diseases and with immune response to transplants. Some DNA-based typing techniques have previously been described for MICA including sequence-based typing (SBT) and analysis of single strand conformational polymorphisms (SSCP). In the present experiments we have developed a strategy that allows identification of all 54 MICA alleles described so far, using group-specific polymerase chain reactions (PCR) and sequence-specific oligonucleotide probes (SSOP). To analyze for the polymorphisms in exons 2, 3, and 4 an initial screening with group-specific primers, based on polymorphism at position 69 of exon 2, and at position 615-616 of exon 4, was used to determine four major groups of alleles. Then group-specific PCR amplifications were performed and the amplified DNA was hybridized with the corresponding panels of SSOP. An additional amplification was performed with locus-specific primers and hybridized with a set of SSOP to identify and/or confirm the presence of some of the alleles. Unequivocal MICA typing was achieved for 97 of 103 individuals. Of 54 previously described alleles, only 14 were observed in this population. One unexpected hybridization pattern was observed, and molecular cloning and sequencing confirmed it to be a novel sequence, which was given the local designation MICA-055D. The gene frequencies among 103 unrelated North American Caucasian donors were determined and the linkage disequilibrium between MICA and HLA-B was analyzed.

Enzyme-linked DNA oligotyping. A practical method for clinical HLA-DNA typing.

The use of PCR and oligonucleotide hybridization has increased the accuracy and resolution of typing for HLA class II alleles, but current procedures, performed in batches, take too long and are not suited for testing single samples. We have developed a typing method using enzyme-linked oligonucleotides and PCR products immobilized in 96-well trays. Trays preloaded with typing probes, covalently linked with alkaline phosphatase, have been kept for weeks at 4 degrees C without loss of enzyme-probe activity. Bound alkaline phosphatase was detected using a color reaction with enzymatic amplification which produces readings in 30 minutes. Coupled with a quick DNA preparation method, results can be obtained in about 4 hours. This method can be easily performed in small laboratories. It is accurate, reproducible, and sensitive, and will make oligotyping for HLA alleles more convenient for testing clinical samples.

New alleles of the HLA-B15 family.

In a previous study of B locus alleles by sequence-specific oligonucleotide probe (SSOP) hybridization, we observed 18 novel patterns in a panel of 360 individuals. Four of these novel patterns were caused by alleles of the human leukocyte antigen (HLA)-B15 group, and three were available for this study. These alleles were found in Oriental, Latin American, African American, and Caucasian individuals. In addition, we analyzed a Caucasian subject who was found by serology to have an unusual B15 specificity. We sequenced these four samples by performing amplification from genomic DNA using polymerase chain reaction primers designed to obtain HLA class I products that included exon 2 and exon 3 as well as the intervening intron. The amplified segments were cloned and identified by colony hybridization with nonradioactive SSOP. Nucleotide sequences were obtained using an automated DNA sequencer. The allele B*1530 differs from B*1501 by a substitution of Asp for Asn in position 114 and Ser for Tyr in codon 116. The new allele B*1531 differs from B*1502 at amino acids 94, 95, and 152. The variant B*1524 was found to have N-77, I-80, A-81, L-82, R-83. A similar motif exists in B locus alleles that have the supertypic specificity Bw4 and in B*1513, B*1516, B*1517, and B*1523; it is likely to have been generated by gene conversion. Finally, the novel allele B*1527 is similar to B*1501 except for the presence of Phe instead of Tyr at position 99. Because this change exists also in B*1506, it is possible that B*1506 was derived from B*1501 through B*1527. It is of interest that a similar substitution (Cys for Tyr at position 99) distinguishes A*0201 from A*0207 and is known to determine an epitope recognized by T cells. Thus, B*1527 may also carry a change that is functionally relevant in cell-mediated immunity.

Multiple epitopes of HLA-DRB1*0411 are recognized by T-cell clones originated from individuals carrying other DR4 subtypes.

HLA polymorphism dictates the binding and recognition of specific peptides, leading to variations in individual immune responses and may contribute to autoimmune disorders and outcome in organ transplantation. We have studied the molecular basis for the cellular recognition of DRB1*0411 in individuals carrying other sequence-related DR4-alleles by characterization of T-cell clones (TLC). A set of 166 TLC were raised by priming cells from DRB1*0401,0402 and DRB1*0405,0901 individuals and 52 of them recognized DRB1*0411. Five distinct patterns of T-cell allorecognition were found: DRB1*0411 alone, DRB1*0411 and 0405, DRB1*0411 and 0406, DRB1*0411 and 0407 and DRB1*0411, 0406 and 0407, depending on responder phenotypes and epitopes recognized by their T cells. A stretch of 30 amino acids on DRB1*0411 from positions 57 to 86 behaves as a functional domain and residues S57, R71, E74 and V86 seem to be crucial in forming immunogenic determinants recognized by these TLC. The knowledge of shared amino acid residues between closely related DR4 alleles, which show similar patterns of recognition by T cells could also be useful in the selection of prospective donors for clinical transplantation of solid organs or bone marrow.

Novel HLA-B35 subtypes: putative gene conversion events with donor sequences from alleles common in native Americans (HLA-B*4002 or B*4801).

In a study of 523 normal subjects of differing ethnic groups, including 189 South American Indians, we have described novel hybridization pattern corresponding to 22 potentially new HLA-B locus alleles. Three of these alleles were subtypes of B35. The locally, assigned alleles, B-3504v, B-3505v, and B-3508v have been sequenced and were officially designated as B*3512, B*3517, and B*3518, respectively. In addition, we determined the nucleotide sequence of another new variant, locally designated B-3509.2. B*3517, was found in 3 individuals (2 Hispanic, 1 Caucasian), it differs from B*3505 by 3 nucleotide substitutions that lead to changes in residues 94, 95, and 103. B*3517 differs from B*3501 in residues 97 and 103. B*3518 was found in 7 South American Indian individuals (6 of 124 Toba Indians, 1 of 18 Pilaga Indians). It differs from B*3509 by 2 silent nucleotide substitutions and by one nonsynonymous substitution in codon 156 (Arg-->Leu). B*3512 differs from B*3504 by 3 nucleotides, one of them leading to a substitution in residue 103 (Val-->Leu). B*3509 was observed in 3 individuals from the Wichi tribe. The nucleotide sequence of one of these was determined and was found to differ from B*35091 by two synonymous nucleotide substitutions. The distinguishing amino acid substitutions in residues 95, 97, and 156 contribute to the structure of specificity pockets F, C, and E, and D and E respectively, therefore, it is possible that some of the new alleles may have different peptide binding profiles. It has been shown that differences at residue 156 may elicit different allorecognition and mediate graft-versus-host disease and rejection in bone marrow transplantation. The mechanisms for the generation of these novel alleles may involve gene conversion events in which short exon-3 segments from the common Native American alleles B*4002 or B*4801 were inserted in HLA-B35 backbone structures. The novel allele B*3518 is closely related to B*35092 and to B*3508. Two alternative hypotheses for its generation can be suggested, the most plausible one would involve B*35092, the putative progenitor of B*3518, since both alleles are prevalent in the same Indian tribes.