Identification of the Novel HLA-DPA1*02:02:21 Allele by PolyseqOne and Oxford Nanopore Sequencing.

679 I T W AS SHO W N that subtype B variants of human immunodeficiency virus type 1 (HIV-1) from intravenous drug users (IDUs) differ from the variants of homosexual men in several genomic regions, including vpu, vpr, and sequences encoding the gp120 V3 loop. The most conserved was found to be a synonymous nucleotide substitution in the second glycine codon at the tip of the gp120 V3 loop (GGC). Several epidemiological studies were done among the different risk groups in various European countries and the United States. GGC viruses have been found in 85% of IDUs in northern Europe and in 45% of U.S. IDUs. The GGC pattern was not found in V3 sequences obtained from homosexual men in various European countries and the United States. No epidemiological data on this topic had been gathered in Greece, where the majority of HIV-1 infections are observed among individuals infected sexually and only 4.3% of the cases are observed among IDUs. The predominant HIV-1 subtype in Greece is subtype B. To investigate the situation in Greece, we obtained sequences of HIV-1 RNA encoding the V3 region of the envelope glycoprotein gp120 from 18 individuals infected between 1991 and 1997. Seven of them were IDUs, five were homosexuals, three were heterosexuals, two were bisexuals and for one the root of transmission was unknown. Nested polymerase chain reaction (PCR), direct sequencing, and phylogenetic analysis were performed as described earlier. It was found that five of the seven (71.4%) IDUs had GGC viruses, whereas the other two had non-GGC viruses (Fig. 1). None of the individuals in the other risk groups carried GGC viruses (data not shown). In addition, four of the seven Greek IDUs (57%) had a synonymous nucleotide substitution (TCC) at env position 837, like this Dutch IDUs (Fig. 1). All of the individuals in the other risk groups presented a TCT pattern. Moreover, it was shown that a risk group-associated amino acid signature pattern was observed at env codon 288, where 90% of the sequences from homosexual Dutch men had a threonine residue (ACC) compared with 25% of the sequences from Dutch IDUs. From our results it was found that four of five (80%) homosexual men carried sequences with this signature pattern compared with one of seven (14%) IDUs (Fig. 1). These data may indicate that the HIV-1 epidem ic among IDUs was established from a different source than in individuals of other risk groups, perhaps originating from IDUs of other European countries or from the United States. The GGC pattern has been observed among 41% of subtype B sequences from heterosexuals in The Netherlands, suggesting that a number of heterosexuals were infected from IDUs. Further studies are needed to understand the epidemiology of HIV-1 in Greece. Genbank accession numbers of the sequences described in this article are as follows: AJ224947±AJ224949, AJ224951± AJ224956, and AF094522±AF094530.

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

To establish an epidemiological link between HIV-1 epidemics in U.S. and European homosexual men and intravenous drug users (IVDUs) we analyzed the HIV-1 gp120 V3 sequences in both risk groups. Signature pattern analysis revealed that the V3 sequences of viruses from IVDUs in Northern Europe are distinguishable from those of homosexual men on the basis of one amino acid and two synonymous nucleotide substitutions, which the most conserved was a synonymous nucleotide substitution in the second glycine codon at the tip of the gp120 V3 loop (GGC). This substitution was seen in 17 of 20 (85%) viruses of IVDUs in Northern Europe, in none of 41 homosexual men in either Europe or the United States, and in 5 of 11 (45%) U.S. IVDUs sequences analyzed. Subsequent phylogenetic and multivariate principal coordinate (PCOORD) analyses showed that 16 of 20 (80%) of the Northern European IVDU sequences clustered together with the 5 U.S. IVDU sequences carrying the GGC substitution and away from the sequences of homosexual men from either Europe or the United States. Taken together with the higher level of heterogeneity of U.S. IVDU sequences compared to the Dutch IVDU sequences taken at the same time, these data present suggestive evidence for a U.S. instead of a European origin of the AIDS epidemic among Northern European IVDUs.

HLA-B*14:02:16 differs from B*14:02:01:01 by a synonymous nucleotide substitution in codon 141.

HLA-DQB1*06:03:47 differs from HLA-DQB1*06:03:01 by one synonymous nucleotide substitution in codon 158 in exon 3.

HLA-C*01:02:84 differs from HLA-C*01:02:01:01 by one synonymous nucleotide substitution in codon 48.

HLA-B*58:01:43 differs from HLA-B*58:01:01:01 by one synonymous nucleotide substitution in codon 197.

Two simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions are presented. Although they give no weights to different types of codon substitutions, these methods give essentially the same results as those obtained by Miyata and Yasunaga's and by Li et al.'s methods. Computer simulation indicates that estimates of synonymous substitutions obtained by the two methods are quite accurate unless the number of nucleotide substitutions per site is very large. It is shown that all available methods tend to give an underestimate of the number of nonsynonymous substitutions when the number is large.

HLA-F*01:31 differs from HLA-F*01:04:01 by one nonsynonymous nucleotide substitution in codon 183 in exon 3.

HLA-G*01:62 differs from HLA-G*01:04:01 by one nonsynonymous nucleotide substitution in codon 269 in exon 4.

HLA-G*01:64 differs from HLA-G*01:04:01 by one nonsynonymous nucleotide substitution in codon 107 in exon 2.

To study the molecular epidemiology of HIV-1 subtype B amongst heterosexually infected individuals in The Netherlands. The study population comprised 54 individuals infected by subtype B viruses through heterosexual contacts. Serum samples were collected between 1988 and 1996. Sequences of the gp120 V3 region were obtained from serum samples and analysed by using the signature pattern and phylogenetic methods. In 22 (41%) out of 54 subtype B sequences from heterosexually infected individuals, the synonymous nucleotide substitution in the second glycine codon at the tip of the V3 loop (the GGC pattern), previously identified as specific for Dutch injecting drug users (IDU), was found. The other previously described IDU sequence patterns were observed significantly more often among GGC- than among non-GGC-containing sequences. In addition, we identified another amino-acid change specific for the GGC sequences. In the phylogenetic and principal coordinate analyses, the GGC sequences from heterosexually infected individuals clustered separately from the non-GGC sequences and together with the IDU consensus sequence. Both the nonsynonymous and particularly the synonymous distances amongst the GGC sequences were significantly lower than amongst the non-GGC sequences. Our data provide evidence for a common origin of the viruses in Dutch IDU and the GGC viruses in heterosexuals. We suggest that a considerable proportion of the viruses in heterosexually infected individuals in The Netherlands may have originated from Dutch IDU.

Rates of synonymous and nonsynonymous substitution were investigated for 24 genes in three Drosophila species, D. pseudoobscura, D. subobscura, and D. melanogaster. D. pseudoobscura and D. subobscura, two distantly related members of the obscura clade, differ on average by 0.29 synonymous nucleotide substitutions per site. D. melanogaster differs from the two obscura species by an average of 0.81 synonymous substitutions per site. Using a method developed by Gillespie, we investigated the variance to mean ratio, or Index of Dispersion, R, of substitutions along the three species' branches to test the fundamental prediction of the neutral theory of molecular evolution, E(R) = 1. For nonsynonymous substitutions, the average R, Ra is 1.6, which is not significantly different from the neutral theory prediction. Only 5 of the 24 genes had significantly large Ra valves, and 12 of the genes had Ra estimates of less than one. In contrast, the Index of Dispersion for synonymous substitutions was significantly large for 12 of the 24 genes, with an average of R(s) = 4.4, also statistically significant. These findings contrast with results for mammals, which showed overdispersion of nonsynonymous substitutions, but not of synonymous substitutions. Weak selection acting to maintain codon bias in Drosophila, but not in mammals, may be important in explaining the high variance in the rate of synonymous substitutions in this group of organisms.

Correlation between the rates of synonymous (silent) and non-synonymous (amino acid-changing) nucleotide substitutions in genes is a widespread and yet unexplained genome-level phenomenon, which is in disagreement with the neutral theory of molecular evolution (Kimura, 1983). Comparison of 7732 orthologous genes of mouse and rat confirms the previously observed correlation between the rates of substitutions in non-synonymous and synonymous nucleotide sites. In rodents, this correlation is primarily caused by tandem substitutions and, in particular, by CpG mutation bias leading to doublet nucleotide substitutions. The nature of correlation between the rates of synonymous and non-synonymous substitutions in seven pairs of prokaryotic genomes is unclear.

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