HLA class I allele (HLA-A2) expression defect associated with a mutation in its enhancer B inverted cat box in two families

doi:10.1016/0198-8859(94)90087-6

Human Immunology

Volume 41, Issue 1, September 1994, Pages 69-73

https://doi.org/10.1016/0198-8859(94)90087-6 Get rights and content

Abstract

The present study shows a very highly diminished HLA-A2 cell surface expression with mendelian segregation in two nonrelated Spanish families. The associated haplotype included Cblank-B38-DRB1^∗1301-DQ6 in both families. cDNA sequence analysis in two members, one of each pedigree, revealed the presence of the commonest HLA-A2 allele (A^∗0201), without repetitive mutations that could indicate inappropriate or inefficient translation. Further, the coamplified 3′-untranslated region sequence was also the same described for HLA-A2. HLA-A transcription frequency by means of cDNA PCR-based cloning experiments and by Northern blotting pointed out a relatively low number of HLA-A2 mRNA molecules compared with the complementary HLA-A allele. 5′-Regulatory region sequences from two low-expressing HLA-A2 nonrelated individuals showed a unique and identical single point mutation at position − 101 (T to C), when compared with all MHC class I alleles sequenced so far. Position − 101 is located in the inverted CAT box associated with the MHC class I enhancer B. The fact that this is an extremely well-conserved position leads us to postulate that this change may be the only responsible for the defective expression of HLA-A2.

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Cited by (48)

Inactivation of a functional HLA-A gene: A 4-kb deletion turns HLA-A*24 into a pseudogene
2010, Human Immunology
Citation Excerpt :
Fifty-one of these alleles are null alleles, which lack a serologically detectable product; three alleles show reduced cell surface expression (HLA-A*02:01:01:02L, A*24:02:01:02L, A*30:14L); and in two cases, the correct expression is questionable (HLA-A*32:11Q, A*23:19Q) [3]. The expression deficiency can be caused by point mutations giving rise to premature stop codons [4-7] deletions, or insertions leading to a frame shift and a premature termination [4,8-13], deletions or mutations causing a structural defect in the molecule [14,15], mutations affecting transcription [16,17] and RNA splicing [4,8,18,19], or hypermethylation leading to gene inactivation [20]. Within the HLA class I region, up to 12 different pseudogenes have been recognized [21].
An unusual haplotype without a detectable human leukocyte antigen (HLA)–A allele by serologic or molecular typing methods segregates in a Caucasian family. Microsatellite analysis and fluorescence in situ hybridization implicated that the deletion encompasses a narrow region. To identify the deleted region, five different fragments in close proximity to HLA-A, known to be highly polymorphic, were amplified and sequenced. The presence of heterozygous sequences in all five fragments of the individuals carrying the haplotype with the HLA-A deletion, indicates that the fragments are not involved in the deletion. Therefore, the 5′ primer from the fragment closest to the centromeric side of HLA-A was combined with the 3′ primer closest to the telomeric side encompassing an 11-kb region. Sequencing revealed that a deletion of 4089 bp was present, located upstream of HLA-A, including exons and introns 1–3 of the HLA gene. Sequence information of the 3′ part of HLA-A, downstream the deletion, identified that the deleted allele originates from an A*24 allele. Although different repeat sequences are present in the region both inside and outside the deletion, no evidence points to a retrotransposon mechanism. The detected partial deletion of HLA-A turns this functional gene into a pseudogene.
Discrimination of HLA null and low expression alleles by cytokine-induced secretion of recombinant soluble HLA
2009, Molecular Immunology
The disruption of disulfide bridges can decrease or abolish the cell surface expression of HLA class I molecules. Such disulfide bridges are formed by cysteine residues between amino acid (aa) positions 101/164 (α₂ domain) and 203/259 (α₃ domain). Sequence alterations in codons 101, 164, 203 and 259 have been observed in eleven HLA-A molecules. All of these variants except of A*3014L and A*3211Q have been reported to result in null expression alleles. In the case of HLA-A*3014L, a transversion at nucleotide position 563 replaces cysteine by serine at position 164 of the mature polypeptide. HLA-A*3014L is not detectable by standard microlymphocytotoxicity assay.
To verify low or non-expression of this allele, we cloned soluble HLA-A*3014L and the reference allele HLA-A*3001 into a eukaryotic expression vector and transfected K562, C1R and HEK293 cells. Expression of soluble HLA-A*3014L and HLA-A*3001 was measured in the supernatants of transfected and untransfected cells incubated with or without IFN-γ and/or TNF-α using a W6/32 and anti-β₂-microglobulin-based sandwich ELISA. Expression of mRNA transcripts of both alleles was determined by real-time RT-PCR.
HLA-A*3014L was not detected in the supernatant of unstimulated transfectants. Stimulation with IFN-γ and/or TNF-α led to an increase of HLA-A*3014L secretion to a detectable level and increased HLA-A*3001 expression up to 8-fold, but did not show any difference in the increase of mRNA levels between HLA-A*3014L and A*3001. Because of this lack of any difference in the mRNA transcription, the protein expression defect is most likely caused by the missing disulfide bond formation in the α2 domain.
Thus, exposing the cells to cytokine stress allows to distinguish between low- and non-expressed alleles and to classify alleles with a questionable expression pattern (Q alleles). Classifying HLA alleles in expressed and non-expressed variants is essential for matching assessments. Additionally, this discrimination between cytokine inducible and non-inducible defect alleles may be important in allotransplant settings in which a cytokine storm usually occurs following pre-transplant myeloablative conditioning or post-transplant immunosuppressive therapy.
Disulfide Bridge Disruption in the α2 Domain of the HLA Class I Molecule Leads to Low Expression of the Corresponding Antigen
2006, Human Immunology
Using sequence-based typing, we have identified a novel human leukocyte antigen (HLA)-A*30 allele, HLA-A*3014L, with a low expression pattern. The sequence of HLA-A*3014L is identical to that of HLA-A*3001 except for a G to C substitution in exon 3 at nucleotide position 563, resulting in an amino acid difference at position 164 (Cys to Ser). Due to the cysteine substitution, a disulfide bridge in the α2 domain of the HLA class I heavy chain cannot be formed. By using the standard microlymphocytotoxicity test, the HLA-A30 antigen cannot be detected. By flow cytometric analysis of the cell-surface expression at either 37°C or 30°C, a temperature-sensitive expression pattern of the HLA-A*3014L antigen was observed. Only by incubating the cells at 30°C, which increases the stability of HLA class I heavy chains, was a weak but clearly detectable HLA-A*3014L expression found. The mRNA expression level of the HLA-A*3014L allele was not affected by the nucleotide substitution. The intrachain disulfide bond formation in the α2 domain is essential for the normal expression of the HLA molecules. Reduced protein expression is probably caused by incorrect HLA class I heavy chain folding and HLA class I complex assembly.
Identification of HLA-A*0111N: A synonymous substitution, introducing an alternative splice site in exon 3, silenced the expression of an HLA-A allele
2005, Human Immunology
A new variant of the HLA-A*010101 allele designated as HLA-A*0111N, previously known as HLA-A*010101var, was identified in a patient requiring a stem-cell transplantation. The patient was typed by serologic methods as HLA-A2 homozygous and by sequence-based typing (SBT) as A*010101,020601. Flow-cytometric (FCM) analysis with 11 human monoclonal antibodies (mAbs) for the A1 molecule confirmed lack of any cell membrane expression of the A*0111N allele. One-dimensional isoelectric focusing (1D-IEF) of total cell lysate from the patient’s cells revealed no cell surface and cytoplasmic A1 protein expression, whereas the HLA-A2 molecule was identified by both FCM analysis and 1D-IEF. DNA sequence analysis showed the presence of a synonymous substitution from G to T at position 597 in codon 175. RNA SBT revealed a deletion of 24 bp in exon 3, position 596 through 619, encoding codons 175 through 182 of the HLA-A*0111N allele. The synonymous substitution introduced a new splice site, resulting in an efficient splicing, because no classical A1 protein could be detected in the patient. This alternative splicing prevented the translation into a correct and stable class I molecule expression on the cell surface.
HLA-DQA1 introns 2 and 3 sequencing: DQA1 sequencing-based typing and characterization of a highly polymorphic microsatellite at intron 3 of DQA1*0505
2005, Human Immunology
DQA1 class II gene encodes the α-chain of the human leukocyte antigen (HLA)-DQ heterodimer. Sequencing-based typing (SBT) for HLA genes is the most powerful methodology described. However, most of the SBT procedures reported for HLA class II genes are not able to define complete exon 2 region. For that purpose, we have characterized introns 2 and 3 from most DQA1 alleles to design amplification procedures that were able to obtain complete exon 2 and 3 sequences from DQA1 genes. This coding information allowed us to reduce the number of ambiguities for DQA1 typing. DQA1 intron 2 and 3 characterization demonstrated the presence of two polymorphisms for alleles with the same exons 2 and 3 sequence from DQA1*05 group. Different samples including the DQA1*050101 alleles showed a single nucleotide polymorphism at position 53 of intron 2 (G53T). Additional haplotypic analysis showed the possible association of T53 allele with the Ax-Cw5-B18-DR17-DQ2 extended haplotype. On the other hand, DQA1*0505 sequencing from different control samples noticed the existence of a microsatellite (TTTC/AAAG)n located at position 126 of intron 3. Fragment length analysis demonstrated a high polymorphism for this short tandem repeat system (0505STR), defining alleles that ranged from 8 to 20 repetitions in our population.
Nomenclature for factors of the HLA system, 2004
2005, Human Immunology

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^☆: The new nucleotide sequence data reported in this article have been submitted to the GeneBank database and have been assigned the following accession numbers: HLA-A2^∗U02935, HLA-A11 U02934, and HLA-A23 U02936.

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HLA class I allele (HLA-A2) expression defect associated with a mutation in its enhancer B inverted cat box in two families☆

Abstract

Immunol Today

Cell

Cell

Association with ß2-microglobulin controls the expression of transfected human class I genes

J Immunol

Association of class I major histocompatibility heavy and light chains induced by viral peptides

Nature

Transcriptional control signals of a eukaryotic protein-coding gene

Science

Cloning and complete sequence of an HLA-A2 gene: analysis of two HLA-A alleles at the nucleotide level

J Immunol

The HLA-Aw24 gene: sequence, surroundings and comparison with the HLA-A2 and HLA-A3 genes

Immunogenetics

Promoter region of HLA-C genes: regulatory elements common to and different from those of HLA-A and HLA-B genes

Immunogenetics

Two upstream elements activate transcription of a major histocompatibility complex class I gene in vitro

Nucleic Acid Res

α-Interferon induced transcription HLA and metallothionein genes containing homologous upstream sequences

Nature

Allele-specific down-regulation of MHC class I antigens in Burkitt lymphoma lines

Cell Immunol

Cis-acting regulatory elements abrogate allele-specific HLA class I gene expression in healthy individuals

J Immunol

Association with ß₂-microglobulin controls the expression of transfected human class I genes