HLA class-I and HLA class-II phenotypic, gene and haplotypic frequencies in Tunisians by using molecular typing data.

The aim of this study is to define a reliable reckoning of gene frequencies and six-locus haplotypic frequencies of HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, HLA-DQB1 and HLA-DPB1 in the Tunisian population. One hundred unrelated random, healthy people originating from various parts of Tunisia were typed for the alleles of the loci mentioned above by using the molecular techniques polymerase chain reaction--hybridization with oligonucleotide probe (PCR-SSO) and sequence specific primers (SSP). The population studied appeared to be in Hardy-Weinberg equilibrium. Allelic frequency distributions were observed at each locus. The most frequent HLA-A alleles were HLA-A*02 (39%) HLA-A*0101 (25%), HLA-A*30 (21%) and HLA-A*2301 (18%). Moreover, HLA-3A*3601, HLA-1A*6601, HLA-1A*3402 and HLA-2A*8001 were found; however, no HLA-A*4301 was detected. For the HLA-B locus, the most common in descending order were HLA-B*44 (22%), HLA-B*5001 (19%), HLA-B*51 (16%) and HLA-B*18 (15%). Among the 28 alleles HLA-Cw detected, HLA-Cw*6 and HLA-Cw*7 were highly predominant with the frequencies of 33 and 30%, respectively. For the HLA class-II loci, HLA-DRB1*0701, HLA-DRB1*11, HLA-DRB1*13 and HLA-DRB1*03 were the most frequent DR alleles. For the HLA-DPB1, HLA-DPB1*0401, HLA-DPB1*0301 and HLA-DPB1*0201 were the most frequent DP alleles. Many haplotypes were in a strong positive-linkage disequilibrium. The most frequent haplotypes for HLA-A, HLA-B, HLA-C and HLA-DRDQ were HLA-A*3301, HLA-B*1402, HLA-Cw*0802, HLA-DRB1*0102, HLA-DQA1*0101 and HLA-DQB1*0501; HLA-A*2402, HLA-B*0801, HLA-Cw*0702, HLA-DRB1*0301, HLA-DQA1*0501 and HLA-DQB1*0201; HLA-A*2902, HLA-B*4403.1, HLA-Cw*1601, HLA-DRB1*0701, HLA-DQA1*0201 and HLA-DQB1*0202; HLA-A*3002, HLA-B*1801, HLA-Cw*0501, HLA-DRB1*0301, HLA-DQA1*0501 and HLA-DQB1*0201, with frequencies between 0.025 and 0.015. These data can be used as control data for HLA disease associations and paternity studies, but they are also important for the evaluation of the probability rate of success in determining the optimal matched donor in unrelated stem transplantation for Tunisian patients or patients of Tunisian origin.

A new HLA-B44 allele (B*44020102S) with a splicing mutation leading to a complete deletion of exon 5.

Using a combination of serology and polymerase chain reaction with sequence-specific primer (PCR-SSP), we have identified in a volunteer bone marrow donor a new HLA class I antigen within the B44 serotype. This human leukocyte antigen (HLA)-B44 variant was typed as 'blank' by microlymphocytotoxicity, whereas the B*44020101 allele was identified by PCR-SSP. A family study confirmed the Mendelian segregation of this blank antigen identified on one of the maternal haplotype transmitted to her child. The DNA sequence of B*44new, now referred to as B*44020102S, performed from the promoter region to the 3' untranslated region revealed a single nucleotide difference (A/G) compared to B*44020101 at the end of intron 4 in the acceptor-splicing site. This mutation leads to an incorrect splicing characterized by the deletion of exon 5 that encodes the transmembrane domain of the HLA antigen. Indeed, full-length complementary DNA sequencing revealed a complete absence of exon 5. Fluorescence-activated cell sorter analysis confirmed the absence of expression of HLA-B44 on the cell surface in the donor, compared to the HLA-B44 positive control. The isoelectric focusing analysis failed to reveal the presence of an HLA-B44 antigen in the donor, showing that no normal HLA-B*44020101 allele was synthesized. The new B*440201010102S allele is a soluble form of B44 without any detectable cell-surface expression. It can thus be considered as a soluble antigen, a form apparently inactive and unfit for antigen presentation.

Sequence of four new HLA-Cw alleles: a possible role of interallelic recombination.

In order to extend our current understanding of HLA-C polymorphism, four new alleles have been cloned and sequenced: Cw*1801 in a donor of mixed origin, Cw*02024 in a Senegalese individual, Cw*1205 and Cw*1604 in European Caucasoid blood donors. HLA-Cw*1801, which most likely results from an interallelic recombination between Cw*0704 and 0401 alleles, was not associated with B*8101, but with either B*4403 or B18. The Cw*02024 allele differs from Cw*02022 by a silent mutation in exon 3. Both Cw*1801 and Cw*02024 appear to be rather frequent in populations of African origin but have not yet been detected in Caucasoids. HLA-Cw*1604 differs from Cw*1601 by two nucleotides at codon 156 leading to a Gln to Trp substitution. This new Cw16 subtype was subsequently identified in three additional unrelated families, all of South-European origin, and presented an unusual association with B*4402 in all cases. HLA-Cw*1205 is a composite allele with the alpha1 domain of Cw*1602 and the alpha2 domain of Cw*1203. It appears to be rare, at least in European Caucasoids. Three of these four alleles may have resulted from gene conversion-like or interallelic recombination events.

Restriction fragment length polymorphism of DQB and DRB class II genes of the ovine major histocompatibility complex.

The ovine major histocompatibility complex (MhcOvar) class II region was investigated by Southern blot hybridizations using ovine probes specific for the second exons of Ovar-DRB and Ovar-DQB genes. Multiple bands were revealed when genomic DNA was digested with each of five restriction enzymes (BamHI, EcoRI, HindIII, PvuII and TaqI), and successively hybridized with the two radiolabelled ovine probes. Restriction fragment length polymorphisms (RFLPs) were analysed in 89 sheep originating from six inbred families and the inheritance of the fragment patterns was determined. Forty-one fragments were recorded with the DQB probe; 32 were detected with the DRB probe. They constituted 9 DQB and 10 DRB allelic patterns. Twelve DQB-DRB haplotypes were resolved in this study.

Apparent HLA DR triplet due to the coexpression of a DRB5-encoded molecule on a DR1 haplotype.

HLA DR1 molecules are coded by a single polymorphic DRB1 gene. We have observed rare DR1 cells in one Caucasoid family and three unrelated individuals that also reacted with some anti-DR2 sera. Since the second DR antigen was normally expressed, these cells appeared as triplets. Contrary to serology, the cells were not typed by HTCs defining Dw2, Dw12, and Dw21. Further investigations on these unusual DR1+2* haplotypes were conducted by DNA oligotyping and by sequencing of the DRB first-domain exon. The results showed that these DR1 haplotypes, besides their DRB1*0101 allele, carried also a DRB5*0101 allele.

Alloantisera can detect some of the variants of HLA DRw13 identified by oligotyping.

Oligotyping has revealed a considerable polymorphism of HLA DRw13; so far, 5 alleles coded by DRB1 have been identified. An even greater number of haplotypes are apparent when considering the association of DRw13 with alleles coded by DRB3 and DQB1. Serology can now define some of these variants which had formerly escaped detection. We have tested by serology and by oligoprobes 66 genotyped Caucasoid cells comprising the known DR and DQ alleles. Among the 28 DRw6 cells, 10 different haplotype patterns were selected, of which 8 concern DRw13 and 2 DRw14. Four variants of DRw13 were represented: DRB1 *1301, *1302, *1303 and *1305 associated with usual and with uncommon alleles belonging to DRB3 and to DQB1. We have centered our analysis on 19 sera of the XIth Workshop defining DRw6. Two sera (639, 826) were noteworthy since they reacted with certain subtypes of DRw13 and with DRB1 *1102, *1103 only, the other DR specificities remaining negative.

HLA-DRw11, DQw7 has four cellular subtypes revealed by homozygous typing cells and undetected by restriction fragment length polymorphism.

HLA-DRw11 is in strong linkage disequilibrium with DQw7. We have investigated the heterogeneity of DRw11, DQw7 by cellular typing and by HLA cDNA probes. The results obtained by local and other homozygous typing cells in four informative Caucasoid families and 20 genotyped individuals demonstrate the existence of at least four different cellular subtypes. The frequency of DRw11, DQw7 is 25.7% with the following distribution of subtypes: Dw5 (17.3%), JAC (4.2%), JVM (2.8%), TIS (0.7%), and blank (0.7%). JVM (10W 9039) has been previously described; TIS (10W 9042) and JAC are postulated new specificities from our laboratory. None of these subtypes typed for DRw11, DQw1 cells. The cellular heterogeneity contrasts with the absence of polymorphism observed by serology and by restriction fragment length polymorphism after hybridization with DRB, DQA, and DQB probes. Variation in amino acids of the DRB1 chain has been reported for at least three variants: Dw5, JVM, and TIS (more recently).

Diversity among DRw13 DQw7 haplotypes as revealed by serology Dw typing and restriction fragment length polymorphism.

HLA-DRw13 is in linkage disequilibrium with DQw6; an unusual association, DRw13 DQw7, is found in 2% of our Caucasoid population. Investigation of genotyped individuals and of families by two allosera and by Dw typing revealed two subtypes: one recognized by homozygous typing cell HAG and by two allosera, the other subtype remained unreactive with both reagents. Analysis of DNA fragments with DNA probes indicated that the HAG-negative subset had DNA fragments in common with DRw6 while the HAG-positive subset shared DNA fragments with DR5. However, all DRw13 DQw7 cells are Dw24 as seen by hybridization with DRB probe.

Extended HLA haplotypes in multiplex families with insulin dependent diabetes mellitus in Tunisian population: HLA serological typing and RFLP analysis.

Haplotypes including HLA A, B, C, DR, and DQ were compared in a study population comprising 18 Tunisian multiplex families with diabetic children. Eighty haplotypes found in IDDM patients were compared with 148 haplotypes present in healthy family members. RFLP analysis showed that two DR subtypes were significantly more common in the diabetic haplotypes (DR4-DQw8: 82 per cent in IDDM members compared to 0 per cent in healthy members, p less than 0.001 and DR-Dw25: 56 per cent in IDDM patients compared to 16.7 per cent in healthy members, p less than 0.001) and these were in most cases found in haplotype combinations with HLA A2 B44 DR4 DRw53 and HLA A 24 B18 DR3 genes, respectively.