Elongation of the cytoplasmic domain,due to a point deletion at exon 7, results in an HLA-C null allele,Cw*0409 N

The development of molecular techniques for HLA typing has allowed the identification of genes previously assigned as serologic blank alleles. Lack or poor cell surface expression has been found for molecules coded by HLA-A, -B, -DRB4, -DRB5, and -DPB1 genes. In this report we describe the first HLA-C gene encoding for a null cell surface molecule. HLA-Cw*0409 N shows a point deletion at position 1095 within exon 7. This mutation provokes a codon reading shift, generating a new translation stop codon 97 bp downstream to that described in alleles normally expressed. This new stop codon location implies the presence of 32 extra amino acid residues in the cytoplasmic domain. Transfection experiments suggest that elongation of the cytoplasmic domain in Cw*0409 N would be the cause of cell surface expression failure, although Cw*0409 N heavy chain is able to create stable complexes with beta2-microglobulin. HLA-C fragment length analysis in a small selected group of samples with B44-Cblk haplotypic associations allowed us to identify two additional subjects showing both a serologic silent Cw*04 allele and a point base deletion at the 3' end of the HLA-C gene. This finding indicates that the allele frequency of Cw*0409 N within serologic C blank alleles would be appreciable, although basically restricted to the (A23)-Cw*0409 N-B*4403-DR7-DQ2 haplotype.     

HLA-C typing of eleven Papua New Guineans: identification of an HLA-Cw4/Cw2 hybrid allele

HLA-C polymorphism of 11 individuals from Papua New Guinea was studied by serology and DNA typing (SSP ARMS-PCR). To resolve certain discrepancies HLA-C alleles were cloned and sequenced. Five alleles were identified by sequencing, four of which; Cw*0304, Cw*0401, Cw*12022 and Cw*1502 have been identified previously in other populations. The fifth allele, which was found in four individuals is a novel HLA-C allele. The new allele, called HLA-Cw*0403 is most similar to HLA-Cw*0401, differing by 10 nucleotides, 9 of which are located in the region from nucleotide 98 to 218. This region of Cw*0403 is identical to both HLA-Cw*0201 and Cw*02022. The 9 nucleotide differences between Cw*0401 and Cw*0403 result in 6 amino acid differences in the α1 domain. These amino acids in Cw*0403 may contribute to the serological typing of some, but not all Cw*0403 expressing cells. The Final difference between Cw*0401 and Cw*0403 is a coding substitution at nucleotide 979 in exon 5. The guanine found in Cw*0403 is identical to all HLA-C alleles except HLA-Cw*0401, which has an adenine. The Cw*0403 allele was most likely formed by a gene conversion event between Cw*02 and Cw*04, involving a minimum of 121 to a maximum of 215 nucleotides.     

HLA-Cw*0409N is associated with HLA-A*2301 and HLA-B*4403-carrying haplotypes

The associations of HLA-B*4402 and HLA-B*4403 with alleles of HLA-A and HLA-Cw were investigated in panels of HLA-B*4403 and HLA-B*4402 homozygous individuals and in selected individuals carrying HLA-Cw*04 and HLA-B*4403. Some of these individuals were genotyped and also carried (HLA-DRB1*0701, DQB1*02). Among the latter, we studied individuals carrying the conserved extended haplotype (CEH) [HLA-Cw*04, B*4403, FC31, DRB1*0701, DQB1*02]. Four different common (HLA-Cw*, B*44) haplotypes were identified that extended to the HLA-A locus: HLA-A*0201, Cw*0501, B*4402; HLA-A*2902, Cw*1601, B*4403; HLA-A*2301, Cw*0401, B*4403; and HLA-A*2301, Cw*0409N, B*4403. We identified eight unrelated examples of the allele HLA-Cw*0409N. HLA-A*2301 was associated with both HLA-Cw*0401 and HLA-Cw*0409N, suggesting that HLA-Cw*0409N may have arisen from a mutation in a CEH. We estimate that approximately 2 to 5 in 1000 Caucasian individuals carry the allele HLA-Cw*0409N, making it one of the most frequent null HLA alleles known to date. Our findings demonstrate the first example of three different HLA-Cw-determined subtypes of a common or CEH carrying a shared HLA-B allele, in this case HLA-B*4403.     

HLA-C sequence based typing: nucleotide analysis from exon 1 through exon 8. Identification of a new allele: Cw*0718

At present, 128 HLA-Cw alleles have been described. Twenty-four of 128 display critical polymorphisms in contributing to allele identification outside exons 2 and 3. As a matter of fact, complete resolution of Cw*030201, Cw*030202, Cw*0409N, Cw*0501, Cw*0503, Cw*070101, Cw*070102, Cw*070401, Cw*0706, Cw*0711, Cw*0718, Cw*120201, Cw*120202, Cw*150501, Cw*150502, Cw*1701, Cw*1702, Cw*1703, Cw*1801 and Cw*1802 alleles requires nucleotide analysis of exons 1, 4, 5, 6 and 7. Moreover, some alleles (Cw*04010101, Cw*04010102, Cw*07020101 and Cw*07020102) showing nucleotide differences outside the coding regions of HLA-C gene (intron 2) have been reported. High resolution sequence based typing (SBT) developed in this study involves two DNA amplifications and 12 direct sequencing reactions and allows the analysis of HLA-C polymorphisms from exon 1 through exon 8, including intron 2. This typing procedure identifies all 128 Cw alleles described so far. Nevertheless, a number of ambiguous heterozygous typing results may be expected, this being the major drawback of SBT methods. A total of 201 samples were HLA-C typed using SBT strategy here described. The sequence of exons 6, 7 and 8 of HLA-Cw*070102 allele was elucidated. A novel HLA-Cw*07 allele, Cw*0718, was identified in two samples. Cw*0718 differs from the Cw*070101 allele by a unique nucleotide position within exon 6, resulting in an amino acid substitution at codon 324 (Ala-->Val) in the cytoplasmic region of the molecule.     

Role of HLA-B and C alleles in development of psoriasis in patients from North India

Abstract: The association of HLA antigens with susceptibility for development of psoriasis vulgaris has been studied mostly using serologic methods. A number of different HLA class I antigens have been reported to be associated with predisposition to develop this disease. While Cw6 is the allele most commonly thought to confer risk for psoriasis, a number of HLA-B locus alleles have also been thought to be involved and, recently, in at least three studies, a specific amino acid in the HLA-C heavy chain has been implicated. With the recent development of molecular methods for typing for HLA class I alleles, it has become possible to re-examine this association by performing high resolution typing. In the present study we have investigated 38 psoriasis patients and 84 ethnically and geographically matched controls from North India. They were typed for HLA-B and HLA-C. The results showed the Cw*0602 was the main allele that was increased in this group of patients. The previously reported association with alanine-73 was not found to be significant. Cw*0602 was found in 71% of the patients. B*5701 and B*3701 were also increased but appeared to be secondary to linkage disequilibrium with Cw*0602.     

Characterization of a novel HLA-Cw*02 variant, Cw*0208, in a Caucasian individual

We describe an additional HLA-Cw*02 variant, HLA-Cw*0208, which has been identified in a renal transplant recipient of Caucasian origin (Italy). After performing preliminary serological typing, we analyzed exons 2 and 3 of the HLA-C locus polymorphism by cloning the amplified DNA and using a sequence-based typing method. The new allele differs from Cw*020202 by one nucleotide substitution at nucleotide 61 (G-->A) of exon 2, which translates to a difference of one amino acid at residue 21 (His-->Arg) of the HLA-C heavy chain. We propose that Cw*0208 was generated by a random point mutation in codon 21 from the Cw*020202 allele, or through gene conversion of Cw*020202 with another allele, probably the Cw*1205 and Cw*1602 alleles.     

Molecular cloning and polymerase chain reaction-sequence-specific oligonucleotide detection of the allele encoding the novel allospecificity HLA-Cw6.2 (Cw*1502) in Spanish gypsies

A proposed novel allospecificity, HLA-Cw6.2, has been reported to be commonly found (25%) in Spanish Gypsies. Full-length cDNAs of the allele (Cw*1502) coding for this antigen have been cloned in this study. A simple PCR-SSO method for its detection has been standardized and a correlation with the serologic Cw6.2 phenotype has been established. This specificity has been also detected in the homozygous typing cell RMI. Although the primary structures of Cw*1502 and Cw*0601 are not closely related, they share specific motifs that may account for their serologic cross-reactivity. Two novel HLA-C alleles (Cw*12022 and Cw*0602) are also reported.     

Sequencing based typing for HLA-C. Identification of three new alleles: Cw*0307, Cw*0502 and Cw*0504

HLA-C has been described as a transplantation locus in the unrelated bone marrow transplantation setting, and noticeably the number of mismatches between HLA-A,-B,-DRB1 compatible pairs is considerably high. Sequencing based typing (SBT) is an accurate and efficient methodology utilised in the HLA class I and II allele level of resolution. SBT for HLA-C locus was applied on a sample of 40 HLA-A,B,DRB1,DRB3/4/5,DQB1-compatible bone marrow recipient-donor pairs, and 3 new HLA-C alleles have been found. Cw*0307, well defined by serology as Cw3, showed two amino acid changes at the NK motif 77-80 regarding all described Cw*03 alleles, N77K80 instead of S77N80. Two new Cw*05 alleles were described, Cw*0502 properly typed by serology, and Cw*0504 that behaves as a short antigen. Cw*0502 differed from Cw*0501 by only one nucleotide at exon 3, that generated an amino acid replacement at codon 177, K to E. Cw*0504 differs from Cw*0501 by two clustered amino acid positions (114 and 116) placed at the peptide binding site. The rate of new HLA-C alleles found in this small series evidences a high grade of hidden HLA-C diversity in the Spanish population, particularly in the well-defined serologic specificities.     

Identification of a novel HLA-Cw*08 variant allele, Cw*0824

A novel HLA-C allele, Cw*0824, which was identified from an individual of the Han Chinese, differs from Cw*080101 at codon 222 (GAG > AAG ) in exon 4, which results in an amino acid change Glu222Lys.
In recent years, many human leukocyte antigen (HLA)-C alleles have been identified. Up to date, 23 different Cw*08 alleles have been identified according to the IMGT/HLA Database release 2.25.2 May 2009 (1) . Here, we describe the identification of the novel allele HLA-Cw*0824 that was found during routine high resolution sequence-based typing (SBT) of a Chinese stem cell voluntary donor. The HLA alleles of the donor were typed as A*11, 24; B*15, 46; and DRB1*09, 12.     

Polymorphism of HLA class I genes in the Brazilian population from the Northeastern State of Pernambuco corroborates anthropological evidence of its origin

The allelic distribution of human leukocyte antigen (HLA) class I genes (HLA-A, HLA-B, and HLA-Cw) of the population from the State of Pernambuco in Northeastern Brazil was studied in a sample of 101 healthy unrelated individuals. Low to medium resolution HLA class I typing was performed using polymerase chain reaction-amplified DNA hybridized to sequence specific primers (PCR-SSPs). Twenty allele groups were detected for HLA-A, 28 for HLA-B, and 14 for HLA-Cw. The most frequent alleles were HLA-A*02(0.2871), HLA-B*15(0.1238), and HLA-Cw*04(0.2277), and the most frequent genotypes were A*02/A*02(0.0990), B*15/B*15(0.0594), and Cw*04/Cw*04 and Cw*07/Cw*07, both with a frequency of 0.0792. The observed heterozygosity for the studied loci was 79.21% for HLA-A, 87.13% for HLA-B, and 77.23% for HLA-Cw. The most frequent haplotype was A*02-Cw*04-B*35(0.0485), which is also present in Western European, Amerindian, and Brazilian Mulatto populations, but absent in African populations. Taken together, these data corroborate the historic anthropological evidences of the origin of the Northeastern Brazilian population from Pernambuco.     

Characterization of a new HLA-C allele in a Chinese family by sequence-based typing: HLA-Cw*0348

The novel human leukocyte antigen (HLA)-Cw*0348 allele was identified by sequence-based typing in a Chinese family. This allele shows that the sequences of exons 1–3 of HLA-Cw*0348 are identical to those of HLA-Cw*030401 except for a nucleotide substitution that changes CCG to CTG at codon 57, resulting in an amino acid change from Pro to Leu in the protein, and this is a unique nucleotide change among the HLA-C alleles, suggesting a point mutation mechanism. The extended haplotype carrying the new allele was deduced from the family group typing and defined as A*110101, B*1301, Cw*0348, DRB1*0405, and DQB1*0402.     

Novel HLA-Cw*06 allele, Cw*0612, identified by sequence-based typing

In this report, we describe the identification of an human leucocyte antigen-Cw*06 (HLA-Cw*06) nucleotide sequence variant, a new HLA-Cw*0612. The new allele was detected during routine HLA typing by high-resolution sequence-based typing. Allele Cw*0612 showed one nucleotide difference with Cw*0602 at codon 153 (GCG-->ACG) resulting in an amino acid change from alanine to threonine.     

Anchored PCR cloning of the novel HLA-Cw*0704 allele detected by PCR-SSP

Abstract: The novel HLA-Cw*0704 allele, previously detected as the PCR-SSP variant Cw7/8v, has been cloned and sequenced from the homozygous typing cell KR03/4 after amplification by anchored PCR. The nucleotide sequence of Cw*0704 is closely related to those of other Cw*07 alleles, but carries specific changes in exon 3 consistent with its serological behavior - a short Cw7 cross-reactive with antibodies directed against HLA-Cw8. Some of the substitutions of Cw*0704 have not been previously described for HLA-C but are found in HLA-B alleles and in published C sequences of non-human primates. The new allele carries a novel polymorphism in its 5' untranslated region (5'ut) that could be shared by all Cw*07 alleles. By PCR-SSP, Cw*0704 is a relatively common allele in English Caucasoids at a frequency of 4.6%. It is most often observed on HLA-B44 haplotypes previously described as HLA-C "blank", although linkage disequilibria with other HLA-B specificities have been found.     

Molecular diversity of HLA-Cw alleles in the Maratha community of Mumbai, Maharashtra, western India

Recent advances suggest a significant role for the HLA-C locus as a target of alloreactions after bone marrow transplantation. The biological importance of products of the HLA-C locus, both as transplant antigens and as ligands for natural killer (NK) cells, is well established. A total of 10 different serologically defined HLA-Cw antigen specificities (Cw1-Cw10) are encoded by the C locus; however, there are now 151 different alleles that can be identified by molecular methods. Serological definition of Cw alleles therefore includes 20-50% blanks, which cannot be detected by the available antisera. We used the molecular method of polymerase chain reaction (PCR)-based sequence-specific amplification and probe hybridization to define Cw alleles in 91 individuals from the Maratha community, and compared the data with data for 92 serologically typed Maratha individuals from India. We identified Cw*12, Cw*14, Cw*15, Cw*16 and Cw*18, along with the serologically identified Cw*01, Cw*02, Cw*03, Cw*04, Cw*06 and Cw*07 alleles. The HLA-Cw blank allele frequency in the Maratha was reduced from 0.5706 to 0.00. Furthermore, by using a molecular technique, it was possible to identify novel allele subtypes, such as Cw*0104, Cw*0203 and Cw*0707, and a high frequency of Cw* 1801 in the Maratha community compared with other Indian and world populations. Our results will have clinical implications in related and unrelated HLA-matched bone marrow transplantation in India.     

HLA-C locus alleles in patients with psoriatic arthritis (PsA).

The aim of this investigation was to compare the frequency of alleles at the HLA-C locus in patients with psoriatic arthritis to that in the disease free population and to relate the HLA genes to disease phenotype in psoriatic arthritis (PsA). Ninety-four consecutive patients seen in the (PsA) clinic between April and July 1996 and 100 disease-free controls had HLA typing performed by both serologic and molecular techniques. Patients' disease was assessed according to a standard protocol. Fisher's exact test was used to compare the frequency of HLA alleles in the patients and controls. HLA-C determined by PCR-SSP decreased the frequency of non-identified, that is, "blank" (or null) alleles to 6% in patients and controls. HLA-Cw*0602 was present in 32 patients and 18 controls: allele frequency of 17% in the patients versus 9% in the controls (p < 0.01). Amongst patients with psoriatic arthritis, those who carried the HLA-Cw*0602 allele had a significantly earlier mean age of onset of their psoriasis (p = 0.003). This study confirms that molecular DNA techniques improve detection of C-locus alleles. The HLA-Cw*0602 is increased amongst patients with psoriatic arthritis compared to controls. The HLA-Cw*0602 is associated with an earlier age of onset of psoriasis.     

Human leukocyte antigen class I and class II allele frequencies and HIV-1 infection associations in a Chinese cohort

China has one of the most rapidly spreading HIV-1 epidemics. To develop a vaccine targeted to specific human leukocyte antigen (HLA) epitopes in this population, allele distribution analysis is needed. We performed low-resolution class I and II HLA typing of a cohort of 393 subjects from mainland China using a polymerase chain reaction with sequence-specific primers (PCR-SSPs). We found 10 class I alleles present in more than 10% of the population: HLA-A*02, HLA-A*11, HLA-A*24, HLA-B*13, HLA-B*15, HLA-B*40, HLA-Cw*03, HLA-Cw*07, HLA-Cw*01, and HLA-Cw*06. Several class II alleles were found at high frequency (>or=10%): HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DRB1*0701, HLA-DRB1*1501, HLA-DRB1*0401, HLA-DRB1*0901, HLA-DRB1*1201, HLA-DQB1*0601, HLA-DQB1*0301, HLA-DQB1*0201, HLA-DQB1*0501, and HLA-DQB*0303. We also estimated 2- and 3-locus haplotype frequencies. Because this cohort contained 280 HIV-1-seropositive and 113 HIV-1-seronegative individuals, we compared allele and haplotype frequencies between the infected and control groups to explore correlations between HLA antigens and susceptibility/resistance to HIV infection. The HLA-B*14 allele was only found in the HIV-1-seropositive group, and many 2-locus haplotypes were significantly overrepresented in this group: HLA-B*14/Cw*08, HLA-B*51/Cw*14, HLA-A*02/B*13, HLA-A*31/Cw*14, HLA-A*02/Cw*06, and the class II haplotype HLA-DRB1*1301/DQB1*0601. Alleles significantly increased in the HIV-1-seronegative controls were HLA-B*44, HLA-Cw*04, and HLA-DRB1*1402. Overrepresented 2-locus haplotypes in the control group were HLA-B*44/Cw*04, HLA-A*31/Cw*03, HLA-A*03/Cw*07, HLA-A*11/B*13, HLA-A*11/B*38, HLA-A*24/B*52, and HLA-A*11/Cw*01. The 3-locus haplotypes HLA-A*24/Cw*03/B*40 and HLA-A*02/B*15/DRB1*1201 were found to be increased significantly in the control group. These data contribute to the database of allele frequencies and associations with HIV infection in the Chinese population.     

The new HLA-Cw*0442 allele possibly generated by a recombination event involving Cw*04010101 and Cw*1801

A new human leukocyte antigen-C (HLA-C) allele Cw*0442 was identified in a Spanish Caucasian patient by sequencing-based typing (SBT). HLA-Cw*0442 differs from Cw*04010101 by three amino acid replacements at positions 9 (S>D), 11 (S>A), and 14 (W>R).     

Allele-specific amplification of the complete HLA-C gene from genomic DNA - a novel Cw4 allele (C*04:71) with a Cw1 motif in the peptide-binding site

To determine the complete sequence of a newly identified human leukocyte antigen (HLA)-C allele, we designed a method where the full genomic sequence of HLA-C*04 was amplified in isolation from the patient second HLA-C allele in a single polymerase chain reaction (PCR), using primers spanning its 5'- and 3'-untranslated regions. The new allele, officially designated HLA-C*04:71, differs from HLA-C*04:01:01:01 by two single-nucleotide polymorphisms: one determines substitution of phenylalanine for serine 9 at the B pocket of the peptide-binding site; the second substitution is a new polymorphism in intron 5. Phe-9 is characteristic of Cw1 alleles and its presence in C*04:71 most likely affects its peptide-binding repertoire. The principle we have used for C*04:71 isolation could be adapted for unambiguous sequence-based HLA-C typing of selected samples in a clinical setting.     

Characterization of a new HLA-C allele: Cw*1606

HLA-Cw*16 is a relatively common HLA-C specificity among Caucasoids, with Cw*1601 being the most frequent allele. We report herein the identification by sequence-based typing of a new HLA-Cw*16 allele in a Spanish Caucasoid blood donor. The novel allele, designated Cw*1606, differs from Cw*1601 by two nucleotide changes at positions 361 (T to A) and 368 (A to C) in exon 3, which leads to two amino acid changes from Trp (TGG) to Arg (AGG) and from Tyr (TAT) to Ser (TCT) at codons 97 and 99 in the alpha2 domain, respectively. Sequence comparisons suggest that the new HLA-Cw*1606 variant could have arisen from an intralocus gene conversion event.     

Point mutation in the alpha helix of the HLA-C alpha2 domain generates a novel HLA-C allele,HLA-Cw*0106, in a Han Chinese individual in Taiwan

We report herein the identification of a new HLA-C allele using sequence-based typing (SBT). This novel allele, HLA-Cw*0106, was found in a Han Chinese individual from Taiwan. This individual was typed using SBT as having a class I HLA genotype of HLA-A*0206/0207, HLA-B*4601/5601, and HLA-Cw*0102/0106. This new allele differs from HLA-Cw*0102 in one of the nucleotides of the polymorphic exon 3 at codon 152 (GAG-->GTG; E152V). This residue is located in the alpha helix of the HLA-C alpha2 domain and may have the potential to affect the binding of HLA-C molecules with antigenic peptides and/or the interactions with the T cell receptor. This new allele was detected in a few individuals of Han Chinese in Taiwan, but has not yet been observed in the aboriginal populations in Taiwan.