Characterization Of Novel Alleles Research Articles

Characterization of a novel HLA-A*11:335 allele resulting from a rare interlocus recombination involving HLA-A*11:01:01:01/126 and HLA-H*02:07/14/18 alleles with nanopore sequencing, in a volunteer from the China Marrow Donor Program

BackgroundThe major histocompatibility complex (MHC) in humans includes three classical class I loci (A, B, and C), which are important biomarkers for the transplantation of organs and hematopoietic stem cells. In the MHC, polymorphism is known to be extremely high while interlocus recombination is rare. We report a rare interlocus recombination between HLA-A and HLA-H, which was analyzed using next generation sequencing and nanopore sequencing.MethodsIn the sample, the genotypes of HLA-A, B, C, DRB1, and DQB1 were firstly determined using the methods of sequence-specific primer, sequence-specific oligonucleotide, Sanger’s sequencing, and NGS; however, HLA-A could not be phased. Nanopore sequencing was finally utilized to distinguish the sequence of the novel allele.ResultsFinally, the novel HLA-A*11:335 allele was identified as an interlocus recombination involving HLA-A*11:01:01:01/126 and HLA-H*02:07/14/18 alleles; this was mainly achieved by nanopore sequencing.ConclusionsThe identification of the interlocus recombination indicated that nanopore sequencing can be helpful in the characterization of novel alleles with complex rearrangements. Interlocus recombination has been identified as one of the mechanisms involved in the generation of novel HLA alleles.

Identification of eleven novel HLA alleles by sequence based typing (SBT): A∗02:557, A∗11:01:64, B∗40:299, B∗53:37, C∗01:98 N, C∗02:14:02, C∗04:195, C∗08:110, C∗08:111, DRB1∗03:110, DRB∗13:184

Accurate HLA typing and matched donor selection is essential for graft outcome in patients receiving a stem cell transplant. Misidentification of new alleles can result in graft failure or acute graft vs. host disease. This study identifies eleven novel HLA alleles at class I and class II loci. Method Unusual or potentially novel HLA alleles were detected during sequence specific oligonucleotide (SSO) intermediate resolution typing. Characterization of novel alleles was performed by sequence based typing (SBT) using group specific and locus specific primers. Results Eleven novel HLA sequences were identified and characterized in 14 individuals, including HLA-A∗11:01:64 in a patient and a haploidentical sibling, C∗04:195 in a patient and a fully matched sibling, and DRB1∗03:110 in two haploidentical sibling donors. Nine of the eleven novel HLA alleles resulted in an amino acid substitution. The null allele C∗01:98 N resulted in a premature stop codon in exon 2. Six out of the eleven novel alleles have nucleotide/amino acid changes in exon 4 and the rest in exon 2. Table 1 shows the differences of nucleotides and amino acids between these novel alleles and their most homologous allele and the location of the changes. Discussion Identification and characterization of novel HLA alleles is important for the selection of appropriate stem cell transplant donors aimed at improving graft outcome and patient survival. Download : Download full-size image

The serotonin transporter gene-linked polymorphic region: Identification and functional characterization of novel alleles

of Adult T cell lymphoma 4, number of myelodysplastic syndrome 9, number of macroglobrinemia 3. In NHL, kind of agents is rituximab 135, CHOP regimen 118, COP regimen 13, bendamustin 10. In multiplemyeloma, kind of agents are bortezomib 59, zolendronate 24. In breast cancer, number of CMF therapy 8, number of vinorelbine 6, number of trastuzumab+docetaxel 7, number of eribulin 11, number of number of trastuzumab+vinorelbine 5, number of trastuzumab 1, number of zolendoronate 2, number of LSG15 regimen 2, number of azacitidine 9. The reason of canceled cases, mainly neutropenia, few cases infection. There were no patients who canceled by nausea and vomiting. Key conclusion.– Elderly patients can receive chemotherapy the same as adult patients.

Characterization of Novel Alleles of Toxin Co-Regulated Pilus A Gene (tcpA) from Environmental Isolates of Vibrio cholerae

Vibrio cholerae is causative agent of life threatening diarrheal disease, cholera. The toxin co-regulated pilus (TCP) is a critical colonization factor of V. cholerae and it also serves as receptor for CTXФ. In this study, we describe nucleotide sequence of four novel alleles of tcpA gene from toxigenic and non-toxigenic V. cholerae isolated from environmental sources. The phylogenetic analysis of tcpA revealed that it is related to tcpA of newly emerged O1 strain and unrelated to tcpA of wild type (classical and El Tor strains). All strains showed variant tcpA and also harbored intact Vibrio Pathogenicity Island (VPI). The expression of all variant alleles was demonstrated by RT-PCR.

Characterization of Novel Alleles of the Escherichia coli umuDC Genes Identifies Additional Interaction Sites of UmuC with the Beta Clamp

Translesion synthesis is a DNA damage tolerance mechanism by which damaged DNA in a cell can be replicated by specialized DNA polymerases without being repaired. The Escherichia coli umuDC gene products, UmuC and the cleaved form of UmuD, UmuD', comprise a specialized, potentially mutagenic translesion DNA polymerase, polymerase V (UmuD'(2)C). The full-length UmuD protein, together with UmuC, plays a role in a primitive DNA damage checkpoint by decreasing the rate of DNA synthesis. It has been proposed that the checkpoint is manifested as a cold-sensitive phenotype that is observed when the umuDC gene products are overexpressed. Elevated levels of the beta processivity clamp along with elevated levels of the umuDC gene products, UmuD'C, exacerbate the cold-sensitive phenotype. We used this observation as the basis for genetic selection to identify two alleles of umuD' and seven alleles of umuC that do not exacerbate the cold-sensitive phenotype when they are present in cells with elevated levels of the beta clamp. The variants were characterized to determine their abilities to confer the umuD'C-specific phenotype UV-induced mutagenesis. The umuD variants were assayed to determine their proficiencies in UmuD cleavage, and one variant (G129S) rendered UmuD noncleaveable. We found at least two UmuC residues, T243 and L389, that may further define the beta binding region on UmuC. We also identified UmuC S31, which is predicted to bind to the template nucleotide, as a residue that is important for UV-induced mutagenesis.

A Novel Variant B Allele of the ABO Blood Group Gene Associated with Lack of B Antigen Expression

Background: The gene locus for the ABO blood group system encodes a glycosyltransferase. Alterations in the DNA sequence are associated with the blood groups and the expression levels of antigens on red blood cells. A number of ABO alleles have been described as the molecular basis of weak A or B antigens. Patients and Methods: Here, we describe a novel variant B allele in a blood donor with discrepant results in routine forward (group A) and reverse (very weak anti-B isoagglutinins) ABO blood grouping. Results: Determination of the ABO genotype using polymerase chain reaction-sequence-specific primers (PCR-SSP) indicated blood group A2B. Sequencing of the ABO gene exons 6 and 7 showed for 1 allele a G insertion into the GGGGGG sequence at position 811–816 of exon 7. The 816insG mutation (designated ABO*Bw20) led to a frame shift of the coding sequence and subsequent alteration of the protein sequence. The location of the mutation on a B allele was proven by PCR-SSP. Screening for the novel mutation in 211 blood donors with regular ABO phenotypes indicated that *Bw20 is a rare variant. Conclusions: The low levels of anti-B isoagglutinins associated with this novel variant indicate that residual undetectable amounts of B antigen may be present on red blood cells. The serological and molecular analysis of members of the blood donor’s family further proved the phenotype-genotype correlation of the *Bw20 allele with antigen·and individually variable levels of anti-B isoagglutinins. The characterization of novel alleles associated with ABO subgroups may ensure the correct determination of blood groups in which serological methods are combined with molecular genetic approaches.

Four novel defective alleles and comprehensive haplotype analysis of CYP2C9 in Japanese

Genetic variations in cytochrome P450 2C9 (CYP2C9) are known to contribute to interindividual and interethnic variability in response to clinical drugs such as warfarin. In the present study, CYP2C9 from 263 Japanese subjects was resequenced, resulting in the discovery of 62 variations including 32 novel ones. In addition to the two known non-synonymous single nucleotide polymorphisms (SNPs), Ile359Leu (*3; allele frequency=0.030) and Leu90Pro (*13; 0.002), seven novel non-synonymous SNPs, Leu17Ile (0.002), Lys118ArgfsX9 (*25; 0.002), Thr130Arg (*26; 0.002), Arg150Leu (*27; 0.004), Gln214Leu (*28; 0.002), Pro279Thr (*29; 0.002) and Ala477Thr (*30; 0.002), were found. Functional characterization of novel alleles using a mammalian cell expression system in vitro revealed that *25 was a null allele and that *26, *28 and *30 were defective alleles. The *26 product showed a 90% decrease in the Vmax value but little change in the Km value towards diclofenac. Both *28 and *30 products showed two-fold higher Km values and three-fold lower Vmax values than the *1 allele, suggesting the importance of Gln214 and Ala477 for substrate recognition. Linkage disequilibrium and haplotype analyses were performed using the detected variations. Only five haplotypes (frequency >0.02) accounted for most (>87%) of the inferred haplotypes, and they were closely associated with the haplotypes of CYP2C19 in Japanese. Although the haplotype structure of CYP2C9 was rather simple in Japanese, the haplotype distribution was quite different from those previously reported in Caucasians and Africans. Taken together, novel defective alleles and detailed haplotype structures would be useful for determining metabolic phenotypes of CYP2C9 substrate drugs in Japanese and probably Asians.